Jochen Wuerges

Silvano Geremia
email: sgeremia@units.it

Lucio Randaccio

S L Hubbard

J M Thornton

Structural study on ligand specificity of human vitamin B12 transporters

Keywords: DMB, 5,6-dimethylbenzimidazole, Cba, cobamide, Cbi, cobinamide, Cbl, cobalamin, cobalamin, transport proteins, transcobalamin, haptocorrin, intrinsic factor, comparative modeling, structure comparison

IF, intrinsic factor; HC, haptocorrin; TC, transcobalamin; rms, root mean square

INTRODUCTION

The transport of the essential micronutrient cobalamin (Cbl, vitamin B 12 ) from food to cells relies on three successive proteins in mammals, haptocorrin (HC), gastric intrinsic factor (IF) and transcobalamin (TC) [START_REF] Nexø | Cobalamin binding proteins. In: Vitamin B 12 and B 12 -proteins[END_REF][START_REF] Alpers | Intrinsic Factor, Haptocorrin, and their Receptors[END_REF][START_REF] Rothenberg | Transcobalamin II[END_REF]. Cbl is required by cells as the basis for two enzyme cofactors, methyl-Cbl for methionine synthase and 5'-deoxyadenosyl-Cbl (Ado-Cbl) for methyl-malonyl-CoA mutase [START_REF] Banerjee | The many faces of vitamin B12: Catalysis by cobalamindependent enzymes[END_REF]. Upon initial uptake of Cbl from food, Cbl becomes bound in the stomach to salivary HC. After proteolysis of HC in the duodenum, Cbl is passed on to IF. Mucosal cells in the ileum absorb the IF-Cbl complex via endocytosis mediated by a specific receptor. In the enterocyte, the IF-Cbl complex is degraded and Cbl is transferred to TC which delivers Cbl to cells via the blood [START_REF] Quadros | Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood[END_REF]. Only the fraction of Cbl bound to TC is taken up via endocytosis by a specific receptor on most cell types. HC that is also present in plasma cannot facilitate cellular uptake of Cbl with the exception of hepatocytes. TC-Cbl is degraded in lysozomes to release Cbl for further conversion into the important cofactors. Cbl-deficiency as a result of impaired intestinal absorption is more common among the elderly population and may lead to disorders including hematologic abnormalities and defects in the nervous system and metabolism [START_REF] Wolters | Cobalamin: a critical vitamin in the elderly[END_REF].

Table 1 summarizes some properties of the human Cbl-transporters. Investigation of their gene structure suggested that the three proteins have a common evolutionary origin and that TC has diverged first from the ancestral gene [START_REF] Li | Genomic structure of human transcobalamin II: Comparison to human intrinsic factor and transcobalamin I[END_REF]. The homologous Cbl-transporters show about 25% overall sequence identity but several shorter stretches possess considerably higher similarity. Glycosylation is observed for IF and to an even higher extent for HC, but not for TC [START_REF] Alpers | Intrinsic Factor, Haptocorrin, and their Receptors[END_REF]. The proteins have extraordinary affinity for the vitamin, with similar K d values < 1 pM [START_REF] Fedosov | Comparative analysis of cobalamin binding kinetics and ligand protection for intrinsic factor, transcobalamin, and haptocorrin[END_REF]. However, they show differences in their specificity for Cbl analogues, which are as well produced by microorganisms [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF].

Very recently, the structures of human and bovine TC in complex with Cbl have been solved by X-ray crystallography [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF]. These allowed the detailed description of the Cbl-binding mode and represent the prototype for the family of mammalian Cbl-transporters since no structures have been reported yet for IF or HC. Their structure determination by X-ray crystallography is likely to remain challenging due to difficulties with crystallization of these highly glycosylated proteins. Detailed structural information for IF and HC is of importance for understanding their way of function in greater detail, especially the interactions with the ligand Cbl, the role of glycosylation and the recognition by specific cell surface receptors.

In particular, knowledge of the Cbl-binding mode for all three proteins is essential to rationalize the observed diversity in ligand specificity. IF shows the highest specificity for Cbl and is suggested to select in the first place genuine Cbl among Cbl analogues in the diet, while HC as the least specific binder may remove other Cbl analogues from the circulation that can harmfully compete for the binding site of Cbl in methionine synthase and methyl-malonyl-CoA mutase [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF]. The precise physiological role of HC within the complex process of Cbl internalization is not yet well understood. Furthermore, the structural basis for ligand specificity is of interest in view of the design of Cbl-based bioconjugates. Such bioconjugates show potential as cytotoxic drugs or imaging agents in cancer treatment [START_REF] Russell-Jones | Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours[END_REF] or may function as Cbl-Biochemical Journal Immediate Publication. Published on 5 Feb 2007 as manuscript BJ20061394 competitors to selectively block cell growth in the treatment of AIDS-related lymphoma [START_REF] Pathare | Synthesis of cobalamin-biotin conjugates that vary in the position of cobalamin coupling. Evaluation of cobalamin derivative binding to transcobalamin II[END_REF] or cancer [START_REF] Mclean | Cobalamin analogues modulate the growth of leukemia cells in vitro[END_REF]. These applications are based on the observation that proliferating cells express more surface receptors for holo-TC, and thus take up proportionally more Cbl, than cells in the stationary phase [START_REF] Russell-Jones | Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours[END_REF][START_REF] Amagasaki | Expression of Transcobalamin II Receptors by Human Leukemia K562 and HL-60 Cells[END_REF].

In this study we analyze all three Cbl-transporters regarding structural determinants of ligand specificity. For this purpose, we produced comparative models of Cbl-complexed human IF and HC guided by our recently determined X-ray structures of human and bovine TC. The models are validated with the help of available experimental data for a variety of structural features and are compared to the human TC structure.

METHODS

Modelling

The comparative modelling of IF and HC is based on the structures of human and bovine TC determined by X-ray crystallography (PDB accession codes 2BB5 and 2BB6, respectively [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF]) and on a multiple sequence alignment of IF and HC with these two TC forms shown in Figure 1. This alignment was extracted from a larger multiple sequence alignment to increase its accuracy, employing the following 16 mammalian Cbl-transporting proteins: TC from man (SwissProt entry P20062), chimpanzee (XP_525562), orangutan (Q5REL7), dog (XP_543481), rat (Q9R0D6), mouse (O88968) and cow (Q9XSC9); IF from man (P27352), dog (Q5XWD5), rat (P17267), mouse (P52787) and cow (XP_873322); HC from man (P20061), dog (XP_855361), cow (XP_873306) and hog (P17630). The alignment was performed with CLUSTALW v.1.82 [START_REF] Chenna | Multiple sequence alignment with the Clustal series of programs[END_REF] with the Gonnet 250 scoring matrix. 10 models were calculated both for IF and HC by MODELLER 8v1 [START_REF] Sali | Comparative modelling by satisfaction of spatial restraints[END_REF] using default parameters. The Cbl ligand of the TC template structures were included as rigid body ("block residue" definition in MODELLER) whereas water molecules were excluded from the templates. Conserved disulfide bridges were automatically used as geometric restraints for IF and HC. The best model was chosen according to MODELLER's lowest objective function criterion. The respective best model was subsequently refined iteratively by geometry idealization with REFMAC5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] and model rebuilding with the graphics program O [START_REF] Jones | Improved methods for building protein models to electron density maps and the location of errors in these models[END_REF]. The energy profile (MODELLER's DOPE score) of the models was compared to those of the experimentally determined TC structures. The model geometry was analysed with MOLPROBITY [START_REF] Davis | MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes[END_REF] regarding Ramachandran plot, van der Waals contacts, hydrogen bonds and steric clashes and with PROCHECK [START_REF] Laskowski | PROCHECK: a program to check the stereochemical quality of protein structures[END_REF] regarding amino acid stereochemistry. Cavities in the models accessible to a probe of 1.4 Å radius (corresponding to a water molecule) were found with the program CAVENV [START_REF]The CCP4 suite: programs for protein crystallography[END_REF] and visualized with O. Root mean square (rms) deviations of Cα-atoms in a pair of models were calculated by

Deposition of atomic coordinates

The atomic coordinates have been deposited in the Protein Data Bank (http://www.rcsb.org) with accession number 2CKT for IF and 2CKV for HC.

RESULTS AND DISCUSSION

Description of the IF and HC models and comparison to TC

Comparative models were produced for the human forms of IF and HC on the basis of the sequence alignment with human and bovine TC (Figure 1) and guided by the recently determined crystal structures of human and bovine TC [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF]. The overall correctness of the modelling is indicated by the absence of bad overlaps and large holes. The models show compact globular domains, well defined hydrogen-bonds for buried polar residues and secondary structure elements, as well as extended hydrophobic domain cores.

Small cavities are found which in size and location are similar to the water-occupied cavities observed in the crystal structures of TC. The energy profiles of the models are similar to those of the TC template structures. IF and HC show reasonable stereochemistry with regard to rms deviations from ideal geometry and Ramachandran plot statistics (Supplementary Table 1).

As the template protein TC, the comparative models of IF and HC possess a two-domain architecture in which a larger N-terminal domain ("α-domain"), composed of 12 α-helices in an α 6 -α 6 barrel and a short 3/10 helix, is connected by a flexible linker region to a smaller C-terminal domain ("β-domain") consisting mainly of two β-sheets. The Cbl ligand, with its 5,6-dimethylbenzimidazole (DMB) moiety coordinated to the Co ion ("base-on" form), is tightly enclosed in the domain interface (Figure 2). The sequence identity of human IF and HC as well as the rms deviations of superimposed Cα-atoms of their models are given in Table 2, including also the two TC forms. The regions of elevated similarity between the Cbl-transporters in the sequence alignment of Figure 1 were mapped on the experimentally determined human TC structure. Regarding the α-domain, these regions coincide with the inner six (evennumbered) helices, i.e. the core of the domain, while similarity is spread over the entire β-domain.

An experimental proof of the presence of a two-domain architecture in human IF has been given previously [START_REF] Fedosov | Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin[END_REF][START_REF] Fedosov | Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor[END_REF]. Two fragments, about 30 kDa and 20 kDa in size, were obtained from natural proteolysis of recombinant IF (50 kDa) and closely corresponded to the αand β-domain of TC according to their N-terminal sequencing. Each fragment alone was able to bind Cbl with high affinity and a mixture of both fragments, provided the presence of Cbl, could be assembled into a ternary complex which behaved in a very similar way as uncleaved IF-Cbl regarding Cbl-binding and receptor recognition.

The finding [START_REF] Tang | The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF[END_REF] that truncation of 50 amino acids from the C-terminal end of human IF abolished its Cbl-binding ability is consistent with our IF model since this truncation leads to the loss of all H-bond interactions and most of the hydrophobic contacts between Cbl and the β-domain of IF.

All secondary structure elements of TC are present also in the models of IF and HC (Figure 1). Helices α3, α5 and α13 are shorter in IF than in the other proteins, while particularly extended loops are found between helices α4 and α5 in TC, between α8 and α9 in HC and between strands β1 and β2 in IF.

The flexible linker is three and four residues longer in IF and HC, respectively. Swapping of amino acids is observed between IF and HC in the hydrophobic core of the β-domain, involving the hydrophobic contact Leu 325 -Phe 370 in IF and Phe 335 -Leu 381 in HC. This concerted mutation is located at the same position as that between human and bovine TC which involves a leucine and a valine.

Disulfide bridges

Human and bovine TC show three disulfide bridges in the N-terminal α-domain. In human TC, these are Cys 3 -Cys 249 , Cys 147 -Cys 187 and Cys 98 -Cys 291 . Results from initial multiple sequence alignments indicated that in IF all three disulfide bridges are conserved while for HC, the conservation of only the first two bridges is obtained. The presence of the third bridge, however, appears possible since one of the involved Cys residues (Cys 82 ) is already conserved and the second (Cys 285 ) is shifted away from the conserved position by only one amino acid towards the C-terminus. An alignment featuring the conservation of all three disulfide bridges also in HC can thus be obtained by moving, both for TC and IF, one insertion gap from the nearby linker region to a position N-terminally to the involved Cys residue. A model of HC calculated with the third bridge shows excellent stereochemistry in the loops hosting the bridging Cys residues, i.e. the introduction of a restraint for this third bridge does not lead to distorted backbone geometry.

Apart from the three conserved disulfide bridges, an additional fourth bridge is found in the model of HC, where Cys 365 and Cys 370 connect the β-strands β4 and β5 in the C-terminal domain. No restraint for this bridge was present during modelling and initial refinement. The possibility of the presence of such a bridge was raised earlier based on a primary sequence analysis [START_REF] Fedosov | Sequence, S-S Bridges, and Spectra of Bovine Transcobalamin expressed in Pichia pastoris[END_REF]. A multiple alignment of four known HC sequences (from man, dog, cow and hog) shows that the fourth disulfide bridge is a feature unique to the human form. In conclusion, the following disulfide bridges are present in our models: Cys 8

Glycosylation sites

All glycosylation sites that were predicted on the basis of the Asn-X-Thr/Ser consensus sequence are found to be solvent-exposed in our comparative models (Figure 2). The IF model shows glycosylation sites exclusively on the β-domain, as was confirmed experimentally [START_REF] Fedosov | Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin[END_REF]. Asn 293 was suggested to be glycosylated due to the inability to identify this particular residue during sequencing. Instead, HC also possesses two potential glycosylation sites on the α-domain which fall on the surface region recently proposed as receptor-recognition site of TC [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF]. The presence of carbohydrates on the α-domain of HC, but not on TC, may therefore explain how cell surface receptors for TC discriminate between the two plasma Cbl-transporters TC and HC. Moreover, assuming that the receptor-recognition site is located on the α-domain alone [START_REF] Tang | The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF[END_REF], the lack of carbohydrates on the α-domain of IF may account for the observation that glycosylation does not effect receptor-binding of IF [START_REF] Gordon | Glycosylation is not required for ligand or receptor binding by expressed rat intrinsic factor[END_REF]. Recently, however, the presence of both domains was found to be required for binding of IF to its specific receptor cubilin [START_REF] Fedosov | Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor[END_REF].

Biochemical Journal Immediate Publication. Published on 5 Feb 2007 as manuscript BJ20061394

Cbl environment in IF and HC

The lower side of Cbl (termed α-side) is occupied by the nucleotide moiety and is embedded in the protein (see discussion below). The upper axial side (β-side) contains in our models the ligand H 2 O. This ligand can interact via H-bonds with the main chain oxygen of Pro 149 in IF or Gln 159 in HC. It possesses solvent accessibility which is, however, restricted by the C-terminal part of the loop between helices α7 and α8. The β-side's accessibility to solvent is in agreement with experimental data on kinetics for binding of Cbl with different upper ligands. The fact that very similar binding constants are obtained for Cbl with small ligands, such as CN or H 2 O, and larger ligands such as 5'-deoxyadenosyl, is long considered as evidence that the β-side is at least partly solvent-exposed and thus does not play a role in determining ligand specificity [START_REF] Gräsbeck | Intrinsic factor and the transcobalamins with reflections on the general function and evolution of soluble transport proteins[END_REF]. As concluded in [START_REF] Nexø | Cobalamin binding proteins. In: Vitamin B 12 and B 12 -proteins[END_REF], the transporters show comparable affinities for the various Cbl forms and do generally not discriminate between Cbl with different upper axial ligands (Supplementary Table 2). A special feature in this respect (with unknown physiologic function) may be attributed to TC which treats H 2 O-Cbl differently to CNCbl and N 3 Cbl in that it replaces the H 2 O ligand with a histidine side chain. Spectroscopic studies on the kinetics of ligand exchange at the upper axial position of H 2 O-Cbl upon binding to any of the three transporters [START_REF] Fedosov | Comparative analysis of cobalamin binding kinetics and ligand protection for intrinsic factor, transcobalamin, and haptocorrin[END_REF], revealed reactivity of Co with externally supplied histidine for IF-and HC-bound Cbl, but not for TC-bound Cbl. In fact, the recent Xray structure revealed that upon binding of H 2 O-Cbl to TC, the side chain of a histidine residue forms a coordination bond to the Co ion [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF]. The Co-coordinating histidine in TC is not conserved in IF or HC as is visible from the sequence alignment in Figure 1 and consequently, H 2 O remains coordinated.

Most of the polar and hydrophobic interactions between Cbl and TC are observed also in our comparative models of IF and HC (Table 3). Water-mediated polar interactions are not included in this in HC) is occupied by a Ser 359 and three water molecules. Second, an additional contact exists at the pyrrole ring A of the corrin macrocycle and involves Met 270 in human TC. Third, the phenylalanine side chain is absent at the upper side of the corrin macrocycle between ring C and D. All three transporters have in common the hydrophobic nature of the protein environment in van der Waals contact with the DMB moiety, as has early been concluded from the observation that CN -is unable to displace the DMB from the Co ion in IF [START_REF] Lien | Studies on the Mechanism of Cobalamin Binding to Hog Intrinsic Factor[END_REF].

Compared to the solvent accessible surface of Cbl bound to TC (≈80 Å 2 or 6.6%), the Cbl ligand shows an accessibility twice as high (≈163 Å 2 or 13.3%) when bound to IF and half the accessibility when bound to HC (≈39 Å 2 or 3.2%). In IF, the 5'-hydroxyl group of the nucleotide moiety appears easily accessible to solvent water molecules. The corrin side chains d, e and f as well as an oxygen of the phosphate group possess restricted accessibility while side chain a -c are inaccessible. The same holds for HC where, in addition, side chain f is inaccessible.

Protein Surface Properties

As already found for human TC, no strong electrostatic potential is calculated from the models at the interface of Cbl and either domain of IF or HC, as it can be expected for binding of a neutral ligand (Figure 3). Regarding the domain contact area, a moderate degree of complementarity in the potential appears to exist only for IF which shows the unique feature of a slightly positively charged interface on the α-domain. Instead, the interface surfaces of HC and TC are neutral to slightly negative. Figure 3 illustrates also the shape complementarity between the ligand and the two domains of the transporters. A depression in the otherwise flat interface of the α-domain to the β-domain accommodates about half of the ligand and a small portion of the β-domain which we identified as the turn between strands β3 and β4. In the holo-form of the transporters, this turn is in part wrapped around the nucleotide moiety of Cbl and appears trapped between the α-domain and Cbl. This spatial arrangement can hardly be achieved if Cbl was already bound to the α-domain. As a consequence, it seems necessary for a correct formation of the holo-form that the protein binds Cbl first with its β-domain. Subsequently, the α-domain can attach to the Cbl-β-domain complex and induces (if not already accomplished) the wrapping of the mentioned turn in the β-domain around Cbl's nucleotide moiety. In this latter step, a large amino acid is involved in IF (Trp 348 ) and HC (Trp 359 ), but only a small residue in TC (Ser 359 ). An additional indication for the sequence of Cbl-binding "β-domain before α-domain" is the observation for IF that Cbl binds to the isolated β-domain with higher affinity than to the isolated α-domain [START_REF] Fedosov | Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor[END_REF].

Ligand specificity of the three Cbl-transporters

An early study [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF] demonstrated that the affinity of both IF and TC for 14 different Cbl analogues decreases upon modification of the corrin side chains or the nucleotide moiety. In contrast, HC tolerates major structural differences and even the total lack of the nucleotide moiety. A later study focused on the influence of modifications at the nucleotide moiety on ligand recognition [START_REF] Stupperich | Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin[END_REF]. These assays allowed to file Cbl-binding specificities in the order HC << TC < IF. The likely physiological role thereof implies that the most specific protein IF filters out various Cbl analogues before they pass to the plasma, whereas HC in the blood plasma acts as a scavenger of potentially toxic Cbl-analogues, thus exploiting its lowest level of discrimination among the three transporters.

Attempts to explain the observed differences in specificity have so far been unsuccessful. Earlier, a correlation between His-Co coordination on the upper side of the corrin ring (as is observed for TC) and high ligand selectivity was ruled out by comparative kinetic analysis [START_REF] Fedosov | Comparative analysis of cobalamin binding kinetics and ligand protection for intrinsic factor, transcobalamin, and haptocorrin[END_REF]. The lack of correlation is now corroborated by our finding that the His-Co coordination is present neither in IF nor in HC. Analysis of conservations among the transporters' primary structure, even if supplemented by the detailed knowledge of Cbl interactions with TC [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF], did not suffice to identify crucial residues for ligand specificity. This prompted us to produce and examine comparative models for IF and HC in order to address this issue on the level of tertiary structures.

In the following, our structural analysis of ligand specificity is based on observations from affinity measurements that employ a variety of Cbl analogues [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF][START_REF] Stupperich | Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin[END_REF]. The affinity of a transport protein for a Biochemical Journal Immediate Publication. Published on 5 Feb 2007 as manuscript BJ20061394

given Cbl analogue is expressed as the ratio of the apparent affinity constant to that for CN-Cbl and is determined by an analogue's ability to competitively inhibit the binding of [ 57 Co]CN-Cbl [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF]. The Supplementary Table 2 presents a compilation of investigated Cbl analogues and their affinity relative to CN-Cbl. Modifications can be grouped into those concerning the corrin side chains (a -g) and those concerning the nucleotide arm, in particular the DMB moiety.

The first three analogues in the Supplementary Table 2 involve the replacement of a neutral amide group with a negatively charged carboxyl group at one of the corrin side chains b, d or e (see Figure 4 and the on-line Multimedia adjuncts). Each of these replacements leads in all three protein models to the loss of H-bond interactions to main chain oxygen atoms (Table 3). However, the replacement shows little effect only concerning chain e. A difference between the modification at chain b or d on one hand and chain e on the other hand is the spatial freedom for the latter chain to adapt its conformation according to the introduced electrostatic repulsion while the b-and d-chain are tightly packed against the protein.

Regarding the modification at chain b, the negative charge of the introduced O -suffers from electrostatic repulsion to the backbone oxygens and additionally to two residues in IF (Glu 379 , Asp 383 ), and to only one residue in TC (Asp 393 ) but no other residue in HC. Likewise, the electrostatic surface potential in the contact region of chain d is more negative for IF than for the other two transporters (Figure 3). This can explain the trend of increasing specificity in the row HC < TC < IF regarding these modifications.

Chain d (as also chain c) is fully surrounded by the β-domain alone, i.e. the domain proposed to first accommodate the ligand. Electrostatic repulsion of a modified Cbl will thus disturb the binding process at the earliest step. In contrast, chains b and e are located in the domain interface, and their repulsion will be fully effective only at the subsequent step of formation of the domain interface. This difference is expected to account for the lower affinity of the d-monocarboxylic Cbl-analogue with respect to the band e-forms. Analogue 4 combines the three modifications and confirms the mentioned trend in specificity. The swapping of chain e from below the corrin plane to above (analogue 5) is neither connected to significant loss of affinity nor differences in affinity among the three proteins and, as judged from our models, does not result in steric clash.

The second group of analogues in the Supplementary Table 2 involves modifications at the DMB moiety on the lower side ("α-side") of Cbl. This group can be divided into analogues which preserve the coordination bond between the Co ion and the nitrogen atom N3B of DMB (analogues 6-14 in "base-on" form) and those which do not (analogues 15-19 in "base-off" form). Some analogues were examined in both studies (analogues 7, 10, 12, 16). In the study by Kolhouse and Allen [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF], affinity ratios are systematically lower than those obtained by Stupperich and Nexø [START_REF] Stupperich | Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin[END_REF]. These lower affinities might be due to contamination of protein samples with trace amounts of CN-Cbl or other CN-Cbl analogues, as stated by the authors [START_REF] Kolhouse | Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues[END_REF]. Stupperich and Nexø concluded that modifications on the DMB moiety are tolerated by all binding proteins as long as the Co-DMB coordination is left intact [START_REF] Stupperich | Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin[END_REF]. Upon disruption of this coordination bond, both IF and, to a slightly smaller extent, TC show strongly decreased affinity, in contrast to HC which maintains most of its affinity. Generally, the size of the nucleotide's base had only minor effects on the affinity constants. These conclusions are in full agreement with structural features of our protein models. Smaller modifications at the DMB can be accommodated by the proteins' environment around the nucleotide base (Figure 4 and the on-line Multimedia adjuncts). For base-off analogues, the models suggest that in the final holo-form of the transporter, the nucleotide moiety should approach the Co ion in a way similar to a base-on form in order to avoid major steric clashes.

A striking difference among the Cbl-transporters is the ability of HC to bind with high affinity even those analogues that lack the nucleotide moiety, such as cobinamide (Cbi). In all transporter models, the lower side of Cbl which hosts the nucleotide moiety is surrounded by a region built of the β-hairpin motif β3-turn-β4. It resembles an "arm" at one side of the β-domain as "body" (Figure 4A). HC shows the unique feature of three large residues in van der Waals contact with the nucleotide. Two aromatic side chains, Trp 359 and Tyr 362 , flank the DMB group and the apolar part of the Arg 357 side chain packs against the ribose group (Figure 4B and the on-line Multimedia adjuncts). Trp 359 is present in IF (Trp 348 ) but absent in TC (Ser 359 is at this position). On the contrary, Tyr 362 is present also in TC but absent in IF (Val 351 is found instead). At the position of Arg 357 there is a small polar residue both in IF (Thr 346 ) and in TC (Ser 357 ). These structural differences suggest that the space of the nucleotide moiety of Cbl can be filled upon binding of the nucleotide-lacking Cbi to HC by the side chains of Arg 357 , Trp 359 and Tyr 362 without major main chain rearrangements (Figure 4C and the on-line Multimedia adjuncts). Appropriate side chain conformations of these three residues may enable HC to provide hydrophobic interactions towards the apolar lower side of Cbi and polar contacts towards the solvent. In contrast, IF and TC provide only one aromatic side chain and thus leave a large part of Cbi's apolar lower side accessible to solvent molecules. Major conformational rearrangements in the turn and adjacent amino acids of βstrands 3 and 4 might improve the interaction between Cbi and the hairpin, but remain in any case less optimal than in HC, as is evident from the binding assays. In addition, the positively charged arginine can balance the negative charge at the α-side of cobinamides, stabilizing this Cbl analogue in HC.

We predict that mutations in HC at one or several positions of the three mentioned residues significantly change the binding of Cbl-analogues. Replacing these large residues with smaller residues, such as serine or threonine as present in TC or IF, is expected to deteriorate the packing of the apolar lower side of Cbi to the hairpin motif. Likewise, mutations to render TC and IF equal to HC at the three residues should produce less selective forms of these two Cbl-transporters. In addition, the results of such a study may answer the question whether or not binding of Cbl-analogues without the nucleotide moiety involves structural rearrangements also outside the interface between the analogue and the protein, for instance, in regions determining the flexibility of the hairpin motif.

Binding of Cbl-bioconjugates to TC

In addition to the Cbl analogues compiled in the Supplementary Table 2, binding of Cbl bioconjugates has been studied in great detail for TC [START_REF] Pathare | Synthesis of cobalamin-biotin conjugates that vary in the position of cobalamin coupling. Evaluation of cobalamin derivative binding to transcobalamin II[END_REF][START_REF] Pathare | Synthesis of cobalamin dimmers using isophthalate cross-linking of corrin ring carboxylates and evaluation of their binding to transcobalamin II[END_REF][START_REF] Wilbur | Synthesis and ncaradioiodination of arylstannyl-cobalamin conjugates. Evaluation of aryliodo-cobalamin conjugate binding to transcobalamin II and biodistribution in mice[END_REF][START_REF] Wilbur | Radioiodination of cyanocobalamin conjugates containing hydrophilic linkers: preparation of a radioiodinated cyanocobalamin monomer and two dimmers, and assessment of their binding with transcobalamin II[END_REF]. The effect that the attachment of a molecule to Cbl had on the interaction with TC was systematically investigated in competitive binding assays using Co ion or the 5'-hydroxyl of the ribose moiety had little effect on binding [START_REF] Pathare | Synthesis of cobalamin-biotin conjugates that vary in the position of cobalamin coupling. Evaluation of cobalamin derivative binding to transcobalamin II[END_REF]. These results are rationalized by the three-dimensional structure of human holo-TC [START_REF] Wuerges | Structural basis for mammalian vitamin B 12 transport by transcobalamin[END_REF], showing that sites of conjugation with minor impact on binding are indeed those which suffer less from steric problems with the surrounding protein, i.e. the β-axial position of Co, the 5'-hydroxyl and the side chain e. These locations can accommodate conjugates without restriction on size if an appropriate spacer moiety is inserted between Cbl and the conjugate [START_REF] Pathare | Synthesis of cobalamin-biotin conjugates that vary in the position of cobalamin coupling. Evaluation of cobalamin derivative binding to transcobalamin II[END_REF][START_REF] Pathare | Synthesis of cobalamin dimmers using isophthalate cross-linking of corrin ring carboxylates and evaluation of their binding to transcobalamin II[END_REF][START_REF] Wilbur | Radioiodination of cyanocobalamin conjugates containing hydrophilic linkers: preparation of a radioiodinated cyanocobalamin monomer and two dimmers, and assessment of their binding with transcobalamin II[END_REF].

For the use of Cbl derivatives as therapeutic or diagnostic agents, the correct binding of the derivative to TC is not the only requisite. It is of importance that a complex of TC with a Cbl-derivative shows cellular uptake and intracellular trafficking similar to that of the complex of TC with natural Cbl. McLean et al. investigated the ability of monomeric and dimeric Cbl derivatives to block the growth of leukemia cells [START_REF] Mclean | Cobalamin analogues modulate the growth of leukemia cells in vitro[END_REF]. They found that all those bioconjugates with modifications at the side chain e were good inhibitors of cell proliferation in that they act as competitors with natural Cbl for binding to TC and, at the same time, do not allow coenzyme activity. The constraint that Cbl conjugation should not alter too much the binding affinity to the transport protein holds additionally for IF and HC if the resulting derivative is intended for oral administration. Our comparative models suggest that the functional groups of Cbl can accommodate conjugates in a similar way when bound to IF or TC. In the case of HC, however, available space at the corrin side chains appears more restricted.

In conclusion, this study presented the first structural comparison of the three human Cbl-transport proteins, in particular of their interaction with Cbl and Cbl-analogues. This has permitted a rationalization of biochemical data in the literature about the difference in ligand specificity and the binding of bioconjugates. The protein models indicate the importance of the β3-turn-β4 hairpin motif in the smaller β-domain regarding high affinity of HC for Cbi. Three large amino acid side chains are proposed to supply hydrophobic contacts to the apolar side of Cbi, allowing HC, in contrast to IF and TC, to compensate for the lack of the nucleotide. The position of the hairpin's turn, trapped partially between the α-domain and the nucleotide moiety of Cbl, is suggested to require Cbl-binding first to the β-domain before the compact sandwich complex α-domain-Cbl-β-domain can be achieved. Polar interactions at the corrin side chains of Cbl are very similar in the three proteins; a finding that is consistent with their similar equilibrium dissociation constants. Many interactions of corrin side chains involve the protein backbone and their modification to carboxylic acids causes in general electrostatic repulsion from the tightly packed protein environment. Subtle differences in the proteins' structure may manifest themselves in the kinetics of binding to the different analogues but resolution of such features requires more detailed assays in addition to those assays relative to Cbl available to date. New experimental approaches have recently been presented in the literature [START_REF] Fedosov | Application of a fluorescent cobalamin analogue for analysis of the binding kinetics. A study employing recombinant human transcobalamin and intrinsic factor[END_REF]. The upper diagonal gives sequence identities (relative to the longer sequence in each pair) and, in parenthesis, sequence similarity ("positives" in the substitution matrix of Henikoff and Henikoff [START_REF] Henikoff | Amino acid substitution matrices from protein blocks[END_REF]).

The lower diagonal reports rms deviations of Cα-atoms for equivalenced residues in the models superimposed with DALI [START_REF] Holm | Mapping the protein universe[END_REF]. The number of these residues is given in parenthesis.

human

  -Cys 228 , Cys 125 -Cys 164 , Cys 85 -Cys 270 in IF and Cys 3 -Cys 242 , Cys 132 -Cys 174 , Cys 82 -Cys 285 , Cys 365 -Cys 370 in HC.

  comparison. IF misses the H-bonds to the oxygen atom of the corrin side chains a and f which are present in both TC and HC. Regarding hydrophobic contacts, IF and HC show identical interactions with an exception being the smaller Val 351 in IF at the position of Tyr 362 in HC and TC close to the 5,6dimethylbenzimidazole (DMB) and an additional contact of a threonine C γ atom in IF. TC differs from those hydrophobic interactions in three regions. First, the tryptophane close to DMB (Trp 348 in IF, Trp 359

  [ 57 Co]-CN-Cbl, varying the type and location of the attached conjugate. Attachments at the corrin side chain e showed the smallest decrease in binding (about a factor of 2). Increasingly strong impairment of binding was observed for attachment on side chains in the row b, d and c. Biotin conjugates attached either to the Biochemical Journal Immediate Publication. Published on 5 Feb 2007 as manuscript BJ20061394 © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. Sequence alignment of human and bovine TC with human IF and HC This alignment together with the known three-dimensional structures of the two TC forms were used for comparative modelling of IF and HC. One letter amino acid codes in italics represent secondary structure elements in the α-domain and β-domain, respectively. α-helices (α) and β-strands (β) are numbered consecutively from the N-to the C-terminus. Residues participating in hydrophobic interactions with the ligand Cbl are highlighted in bold. Residues participating in H-bonds to Cbl are marked by the letter for the involved corrin side chain above the residue (those residues employing their side chain are additionally highlighted in grey). The His residue which coordinates to the Co ion of Cbl in human and bovine TC (after displacement of H 2 O at the upper axial position of H 2 O-Cbl) and Cys residues of disulfide bridges are marked by rectangular boxes. Potential Asn-linked glycosylation sites in IF and HC (based on the Asn-X-Thr/Ser consensus sequence) are underlined. See Supplementary Figure 1 for a colour version of this figure.

Figure 2 .

 2 Figure 2. Comparative models of IF and HC (A) Ribbon representation of the IF model showing secondary structure elements, the orientation of the ligand Cbl (magenta) with respect to the N-and C-terminal domain, the disulfide bridges (yellow sticks) and potential Asn-linked glycosylation sites (red). The part of the β-domain coloured in blue corresponds to the 50 C-terminal residues which were removed from the protein in the Cbl-binding studies of Tang et al. [26], see text. (B) Ribbon representation of HC using the colour scheme of panel A. (C) Stereo view of superimposed backbone traces of human Cbl-transporters. The comparative models of IF and HC are shown in green and blue, respectively, and the human TC structure as determined by X-ray crystallography is coloured orange.

Figure 3 .

 3 Figure 3. Surface properties of Cbl transport proteinsThe electrostatic potential (blue, positive potential; red, negative potential) is mapped on the surface of each protein. The proteins are presented here in an artificial "open" conformation in which the α-domain (left) is separated from the β-domain (right) to allow the view on their Cbl-hosting interface. The green line represents the linker region (Figure1). A neutral or slightly negatively potential dominates on all interface regions with the exception of the α-domain of IF, thus possessing moderate charge complementarity. Shape complementarity with the ligand Cbl is much more significant; about half of Cbl's surface is in contact with the α-domain (most of the upper β-side of the corrin ring and side chains a and g) and with the β-domain (most of the lower α-side of the corrin ring and side chains c and d). A region of the β-domain (bordered by a yellow line) wraps around part of the nucleotide moiety on Cbl's α-side and fits into a depression on the α-domain (also bordered yellow). This region corresponds to the turn between β-strands β3 and β4 (see Figure4A); in the holo-form structures, it is trapped between the α-domain and Cbl (bottom scheme). The scheme illustrates the wrapping of the turn around Cbl's α-side at the step of complex formation with the α-domain but this conformational change may be induced

Figure 4 .

 4 Figure 4. Structural analysis of protein-ligand interactions (A) Overview of the ligand position in the transporter with an emphasis on the β-hairpin motif in the βdomain. The ligand (here H 2 O-Cbl) is shown as magenta sticks, the β-strands 3 and 4 as blue ribbons and the rest of the protein with its surface in brown (α-domain) or yellow (β-domain). (B-D) Stereo views of superimposed structures of the transporters in a 5 Å neighbourhood of the ligand (carbon atoms of TC in green, IF in blue, HC in magenta). All labels refer to HC. (B) Interactions of H 2 O-Cbl with the proteins. (C) Interactions of the nucleotide-lacking dicyanocobinamide (Cbl-analogue no. 16 in the Supplementary

Figure 1 TC_hum

 1 Figure 1

TABLE 1 . Properties of Cbl transporters in humans

 1

	Property	IF*	HC*	TC*
	Chromosome	11	11	22
	Source	mainly gastric mucosa	gastric mucosa, white blood	vascular endothelium
			cells, glands, etc.
	Occurrence	stomach, small intestine	body fluids (mainly saliva,	body fluids (mainly
			plasma)	plasma)
	Function	Cbl-transport from proximal	salivary HC: binding of	Cbl-transport from
		intestine to epithelial cells of	dietary HC in the stomach	epithelial cells to tissue
		the ileum	(protection of Cbl from acid	and organ cells
		filtering out Cbl-analogues (?)	hydrolysis and from uptake	Facilitation of Cbl-uptake
			by intestinal fauna ?)	into cells
			plasma HC: Cbl recruitment
			from stores, scavenging of
			Cbl-analogues (?)
	Amino acid	399 (43.4 kDa)	410 (45.6 kDa)	409 (45.5 kDa)
	contents (M W )
	Carbohydrate	9-15 %	30-40%	0
	contents
	(w/w)
	K d (H 2 O-Cbl) †	1 pM	0.01 pM	0.005 pM

TABLE 2 . Comparison of primary and tertiary structures of Cbl-transporters

 2

TABLE 3 . Interactions between Cbl and its human transport proteins

 3

		IF	human HC	human TC	bovine TC
	human IF	-	30% (48%)	23% (38%)	22% (38%)
	human HC	1.3 Å (387)	-	27% (45%)	25% (43%)
	human TC	1.6 Å (379)	1.5 Å (389)	-	73% (81%)
	bovine TC	1.2 Å (381)	1.1 Å (395)	1.2 Å (409)	-

Table 2 )

 2 . Only HC can fill the space occupied by the nucleotide moiety of Cbl after appropriate changes in the side chain conformations of Arg 357 , Trp 359 and Tyr 362 . (D) Environment of a cobamide (here NZA-Cba, Cbl-analogue no. 7) as an example of analogues with small modifications at the DMB moiety. For each panel of this figure, a Multimedia adjunct is provided in the Supplementary Material on-line to facilitate the visualization of the structural features.

© 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 5 Feb 2007 as manuscript BJ20061394 © 2007 Biochemical Society

ACKNOWLEDGMENTS

This work was supported by the Ministero dell'Istruzione dell'Università e della Ricerca, Rome (FIRB2003-RBNE03PX83).


Andre C Stiel

Simon Trowitzsch

Gert Weber

Martin Andresen

Christian Eggeling

Stefan W Hell

Stefan Jakobs
email: sjakobs@gwdg.de

Markus C Wahl

Å bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants

come

INTRODUCTION

Fluorescent proteins, like the green fluorescent protein (avGFP) from Aequorea victoria, used as non-invasive markers have revolutionized cell biology [START_REF] Tsien | The green fluorescent protein[END_REF][START_REF] Lippincott-Schwartz | Development and use of fluorescent protein markers in living cells[END_REF][START_REF] Chudakov | Fluorescent proteins as a toolkit for in vivo imaging[END_REF]. Reversibly switchable fluorescent proteins (RSFPs) are an emerging new class of FPs. These photochromic proteins adopt either a fluorescent (on) or a non-fluorescent (off) state; they may be selectively and reversibly transferred between these two states by irradiation with visible light of appropriate different wavelengths.

The ability to be repeatedly switched on and off is a distinct advantage of RSFPs over the more established, irreversibly photoactivatable or photoconvertable fluorescent proteins. The latter may be activated once from a non-fluorescent into a fluorescent form or their emission spectra may be converted unidirectionally after irradiation with light of appropriate power and wavelength, respectively [START_REF] Lippincott-Schwartz | Development and use of fluorescent protein markers in living cells[END_REF][START_REF] Lukyanov | Innovation: Photoactivatable fluorescent proteins[END_REF]. Thus, only RSFPs will enable repeated measurements of protein movements in single cells, for example. Moreover, RSFPs have been demonstrated to be suitable for attaining spatial resolution far below the diffraction barrier in fluorescence microscopy [START_REF] Hell | Imaging and writing at the nanoscale with focused visible light through saturable optical transitions[END_REF][START_REF] Hofmann | Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins[END_REF].

Likewise they may open a new pathway to high density data storage [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF][START_REF] Sauer | Reversible molecular photoswitches: a key technology for nanoscience and fluorescence imaging[END_REF], applications for which reversibility is important if not mandatory.

Currently, with Dronpa from the coral Pectiniidae [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF], asFP595 (asCP, asulCP) isolated from the sea anemone Anemonia sulcata [START_REF] Lukyanov | Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog[END_REF], and their respective variants, two RSFPs have been described. Both proteins may be repeatedly highlighted, erased, and highlighted again. Up to now, structural information is only available for asFP595. It is an obligate tetramer whose subunits adopt a GFP-like structure, i.e. an 11-stranded -barrel surrounding a central -helix that contains the chromophore [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF][START_REF] Quillin | Kindling fluorescent protein from Anemonia sulcata: dark-state structure at 1.38 Å resolution[END_REF][START_REF] Wilmann | Variations on the GFP chromophore[END_REF]. By using asFP595 protein crystals, in which the chromophores were quantitatively and reversibly photoconverted, it was demonstrated that a cistrans isomerization of the bicyclic chromophore is a key event in the switching process of asFP595 [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF]. As predicted previously [START_REF] Chudakov | Chromophore environment provides clue to 'kindling fluorescent protein' riddle[END_REF], the fluorescent on-state corresponds to the cis conformation, whereas the chromophore in the non-fluorescent off-state adopts a trans conformation. These structural changes are apparently accompanied by different protonation states of the chromophore that determine the fluorescent properties of the protein [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF].

However, Dronpa is currently the RSFP of choice for most cell biology applications, mainly because it is monomeric and because it exhibits a number of favorable spectroscopic properties.

In equilibrium, Dronpa is in its on-state displaying bright green fluorescence with an emission maximum at 518 nm. Its fluorescence quantum yield is high ( FL =0.85) [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF]. For excitation, laser light at 488 nm is conveniently used; intense light of the same wavelength transfers the protein to the non-fluorescent off-state. Subsequent minimal irradiation at around 400 nm transfers the chromophore back to the on-state and restores fluorescence [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF]. Not surprisingly, this protein has already been employed in several studies to monitor the intracellular mobility of fusion proteins [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF][START_REF] Kurokawa | Localized RhoA activation as a requirement for the induction of membrane ruffling[END_REF][START_REF] Fujioka | Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes[END_REF][START_REF] Aramaki | Visualizing neurons one-by-one in vivo: Optical dissection and reconstruction of neural networks with reversible fluorescent proteins[END_REF].

Despite its large potential for cellular applications, little is known about the structural basis of photochromic switching in Dronpa. In this study we have solved the X-ray structure of the fluorescent equilibrium state of Dronpa at 1.8 Å resolution. In the fluorescent equilibrium state, we find the chromophore to adopt exclusively the cis isomeric form. Surprisingly, the chromophore environment of Dronpa is almost identical to that of KikG, a fluorescent protein that does not exhibit photoswitching [START_REF] Tsutsui | Semirational engineering of a coral fluorescent protein into an efficient highlighter[END_REF]. Furthermore, we find structural similarities to asFP595 that point to a cis-trans isomerization of the chromophore as a key event in photochromic switching. Based on this assumption and the obtained structural information we generated rsFastLime (Dronpa-V157G) and Dronpa-M159T, two variants that exhibit strongly accelerated switching kinetics. Our findings support the view of a photoinduced cis-trans isomerization of the chromophore in Dronpa.

EXPERIMENTAL PROCEDURES

Protein production and purification

The expression plasmid pRSETb-Dronpa was a generous gift from A. Miyawaki, Riken Brain Science Institute, Japan. Proteins were expressed in the Escherichia coli strain HMS 174 (DE3) and purified by Ni-NTA affinity chromatography and subsequent size exclusion chromatography according to standard procedures. The purified proteins were concentrated to ~23 mg/ml by ultrafiltration and taken up in 20 mM Tris-HCl, 120 mM NaCl, pH 7.5, for crystallization.

Crystallographic analyses

Dronpa was crystallized without removal of the N-terminal His 6 -tag by sitting drop vapor diffusion at room temperature, by employing a reservoir of 22% (w/v) polyethylene glycol monomethyl ether 3350 and 0.14 M Mg(NO 3 ) 2 . Crystals appeared within two days and continued to grow for several weeks. They could be frozen in liquid nitrogen after transfer into perfluoropolyether. Diffraction data were collected at the PXII beamline of the Swiss Light Source (Villigen, Switzerland) at 100 K using a MarResearch (Hamburg, Germany) CCD detector. The data were processed with the HKL package [START_REF] Otwinowski | Processing of X-ray diffraction data collected in oscillation mode[END_REF].

The crystal structure of the equilibrium state of Dronpa was solved by molecular replacement with MOLREP [START_REF] Vagin | Spherically averaged phased translation function and its application to the search for molecules and fragments in electron-density maps[END_REF] using the structure coordinates of asFP595 (PDB ID 2A50; [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF]) without the chromophore and water molecules. The sequence was manually adjusted and fitted to the 2F o -F c and F o -F c electron densities with COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]. The chromophore was added into an unambiguous and unoccupied patch of the F o -F c electron density. Subsequently, water molecules were placed with Arp/wArp [START_REF] Morris | ARP/wARP and automatic interpretation of protein electron density maps[END_REF]. The water structure was completed manually. Refinement in Refmac5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] was initially restrained by the non-crystallographic symmetry (NCS). In later refinement cycles NCS-restraints were removed. Ultimately, TLS refinement cycles were included, by which individual anisotropic temperature factor corrections for each of the four crystallographically independent molecules were introduced [START_REF] Winn | Use of TLS parameters to model anisotropic displacements in macromolecular refinement[END_REF]. During the entire refinement procedure, 5% of randomly selected reflections were set aside for monitoring of the R free factor [START_REF] Brunger | Assessment of phase accuracy by cross validation: the free R value. Methods and applications[END_REF] (Suppl. Table 1).

Spectroscopic analyses

Absorption spectra were recorded with a Varian Cary4000 UV/VIS Spectrometer. A Varian Cary Eclipse fluorescence spectrometer with excitation at 457 nm (10 nm slit) was used for measuring emission spectra. Within the cuvettes the proteins were irradiated with 405 nm (405 ± 5nm filter) light to quantitatively maintain them in the on-state. We determined the molar extinction coefficients and the fluorescent quantum yields ( FL ) relative to the reported values of Dronpa [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF]. Protein concentrations were measured by using the 280 nm peak of the absorption spectra.

Mutagenesis

Point mutations were inserted by site directed mutagenesis using the QuikChange™ Site Directed Mutagenesis Kit (Stratagene, La Jolla, USA) according to the manufacturers' instructions.

Optical switching

Photoswitching experiments were performed using a modified computer-controlled fluorescence microscope (Leica, Wetzlar, Germany) equipped with a 40×NA 0.6 air objective lens. The microscope was equipped with two 100 W Hg lamps delivering 'blue light' (488/10 nm excitation filter, ~0.3 W•cm -2 ) and 'UV light' (405/10nm excitation filter, ~0.2 W•cm -2 ).

Irradiation was performed in alternate turns for 32 min (Dronpa), 33 s (rsFastLime) or 2.25 s (Dronpa-M159T) with blue light and for 4 s (Dronpa), 2 s (rsFastLime) or 1s (Dronpa-M159T) with UV light together with blue light, respectively. The fluorescence was detected through the same objective lens and recorded by a photomultiplier tube (HR9306-0, Hamamatsu, Hamamatsu City, Japan) using a 500 nm longpass detection filter (HQ 500 LP, AHF Analysentechnik, Tübingen, Germany).

Relaxation kinetics

The determination of the relaxation half-time t 1/2 relax from the off state into the equilibrium was performed as follows: Proteins were expressed in E. coli and the cells were suspended in 1% low melting agarose. 1.5 µl of this suspension were placed in a microtiter well covered by a coverslip and sealed with Vaseline. After complete switching into the off state with 'blue light' (488/10 nm excitation filter, ~0.75 W•cm -2 ) for ~15 min (Dronpa) or ~20 s (rsFastLime and Dronpa-M159T), respectively, the relaxation into the equilibrium state was followed at room temperature (22° C) in the dark by consecutive short measurements with 0.01W•cm -2 blue light (every 10 min, 20 s, or

RESULTS

Crystallization and overall structure of Dronpa on

N-terminally His 6 -tagged Dronpa was expressed in E. coli, purified to apparent homogeneity and crystallized. In solution Dronpa adopts, at thermodynamic equilibrium, the on-state and exhibits a fluorescence emission maximum at 522 nm (Table 1). We find that Dronpa protein crystals display similar fluorescence emission spectra to the protein in solution (Fig. 1A). The fluorescence emission of Dronpa crystals was not further enhanced by irradiating the crystals with light of 405 nm, a wavelength at which off-state Dronpa is converted into the on-state.

Hence we conclude that in the obtained protein crystals Dronpa exclusively adopts the on-state.

We refer to this state as Dronpa on .

Crystals of Dronpa on belong to a centered orthorhombic crystal system with four molecules per asymmetric unit (Suppl. Table 1). The crystal structure was solved by molecular replacement using the structure coordinates of asFP595 (PDB ID 2A50; [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF]) as a search model and omitting the chromophore. The structure was refined at an effective resolution of 1.8 Å maintaining good stereochemistry (PDB ID 2IOV; Suppl. Table 1). R/R free values converged at 18.5/21.6 %.

Residues 2-219 of Dronpa on could be unequivocally assigned in all four copies of the protein and the final models exhibited uninterrupted electron density for the entire main chains and the vast majority of side chains. Only a few surface-exposed side chains were not covered by the final 2F o -F c electron density. The four crystallographically independent molecules adopted highly similar structures (pairwise root mean square deviations [rmsd] 0.15 -0.31 Å for 215 -218 matching C atoms).

We find that Dronpa on exhibits a typical GFP-like fold, comprising an eleven-strandedbarrel with a coaxial, partially helical element between strands 3 and 4, that bears the chromophore (Fig. 1B).

Dronpa on exhibits a slightly non-coplanar cis chromophore

The chromophore is a

[2-(1-Amino-2-mercaptho-ethyl)-4-[1-(4-hydroxy-phenyl)meth-(E)- ylidene]-5-oxo-4,5-dihydro-imidazol-1-yl]
-acetaldehyde moiety that is formed spontaneously from the tripeptide Cys62-Tyr63-Gly64 of the precursor protein (all residue numbering in this manuscript is according to the Dronpa nomenclature; see Fig. 1C). We refer to the chromophore as 'CYG'. The electron density shows unambiguously that in Dronpa on CYG adopts a cis isomeric state (Fig. 2A). The normals to the planes of the five-and six-membered rings span an angle of ~15°. Hence the five and six-membered rings of CYG are slightly non-coplanar.

Since CYG is deeply buried in the protein core, it engages in multiple interactions with neighboring residues. The p-hydroxyphenyl ring of CYG stacks on the His193 side chain and the hydrogen bonding potential of the polar groups is completely saturated (Fig. 2B). The apical hydroxyl group of the p-hydroxyphenyl ring forms a direct hydrogen bond with the side chain of Ser142 and a water-mediated contact with the backbone carbonyl of Glu140. The imidazolinone ring forms an array of seven hydrogen bonds (direct interactions with the side chains of Thr59, Arg66, Arg91, Glu211; water-mediated interactions with the side chains of Gln38, Glu211 and the backbone carbonyl of Asn65). While the central part of the chromophore engages in many polar interactions, van-der-Waals contacts prevail in the peripheral portions of the chromophore.

Thus, the chromophore is firmly attached via its imidazolinone ring but not with its peripheral parts.

An opening to the chromophore

In Dronpa the bulk solvent pH has been shown to elicit a strong influence on its absorption and emission spectra by influencing the protonation state of the chromophore [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF][START_REF] Habuchi | Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa[END_REF][START_REF] Habuchi | Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching[END_REF]. Hence although the CYG resides inside the -barrel, it is not fully shielded from the protein environment. We identified a water-filled opening at the mid-section of the otherwise closedbarrel, between strands 7 and 10 that connects the CYG with the bulk solvent (Fig. 3). The opening possibly relays environmental conditions, such as pH, to the chromophore. It may also serve to discharge harmful oxygen radicals, which are generated at the chromophore, from the immediate chromophore environment. The opening resides within a cleft-like structure between strand 7 and 10 on the surface of the -barrel (indicated by the dotted red line in Fig. 3A,C).

The opening is particularly noticeable in Dronpa on , whereas it is not a general feature of GFP-like fluorescent proteins. However, the cleft-like structure itself is conserved in most GFP-like proteins (Fig. 3C). In fluorescent proteins isolated from Cnidaria species a largely conserved sequence motif (Trp-Glu-Pro) appears to be responsible for a pronounced cleft. This motif, and with it the cleft, are absent in avGFP (Fig. 3B,C).

Remarkably, an opening structurally similar to that identified in Dronpa on has recently been described for the engineered fluorescent protein TurboGFP, which has been derived from a fluorescent protein of an evolutionarily distant marine Copepoda species (Fig. 3) [START_REF] Evdokimov | Structural basis for the fast maturation of Arthropoda green fluorescent protein[END_REF]. In line with our findings, it has been suggested that in TurboGFP the opening facilitates oxygen conveyance to the premature chromophore, thus speeding up maturation [START_REF] Evdokimov | Structural basis for the fast maturation of Arthropoda green fluorescent protein[END_REF].

Structural analysis of the chromophore environments of Dronpa on and asFP595 points to an analogous switching mechanism Dronpa, asFP595 and their derivatives are the only RSFPs described to date [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF][START_REF] Lukyanov | Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog[END_REF]. Besides their color and their tetramerization tendency, the two proteins differ in three obvious properties: First, whereas in Dronpa on the protein backbone is intact, there is in asFP595 a chain break immediately preceding the chromophore. Second, the first chromophoric residues differ in Dronpa and asFP595 (CYG and MYG, respectively). And, third, at thermal equilibrium the Dronpa chromophore adopts a fluorescent cis conformation, whereas the asFP595 chromophore is in a non-fluorescent trans conformation.

Despite these differences both chromophores exhibit a similar stacking interaction between the p-hydroxyphenyl ring and the His193 side chain. The immediate chromophore environments of Dronpa and asFP595 differ only in four residues, i.e. positions 66, 142, 157 and 191. Three of these exchanges (positions 66, 142 and 157) apparently bring about the different isomerization states observed for the two chromphores in their ground states.

In Dronpa on , the hydroxyl group of the p-hydroxyphenyl ring forms a hydrogen bond to the side chain of Ser142 (Fig. 4A,B), thus stabilizing the chromophore in the cis state. In asFP595 position 142 is occupied by an alanine residue that does not facilitate such a hydrogen bond.

Exchange of this alanine residue by serine yields an asFP595 variant in which a substantial proportion of the chromophore population (10-20 %) is at thermal equilibrium in the fluorescent state. Furthermore, structural analysis verified that in asFP595-Ala142Ser the cis position is stabilized by a new hydrogen bond (Fig. 4A) [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF].

If we model a trans conformation of the CYG without adjusting the protein matrix of Dronpa on , it appears that the bulky Val157 and Arg66 sterically hinder the chromophore from adopting a trans conformation (Fig. 5A). In asFP595 the analogous positions are occupied by the slightly smaller residues Ser157 and Lys66 which might explain the lowered energy barrier for the cis-trans isomerization. Furthermore, in ground-state asFP595, Ser157 hydrogen bonds with the apical hydroxyl group of the chromophoric tyrosyl ring and thereby stabilizes the trans conformation. Val157 in Dronpa on does not exhibit the hydrogen bonding capacity to interact similarly with the chromophore. Indeed, exchange of asFP595 Ser157 for valine has been demonstrated to permanently trap the chromophore in the fluorescent cis state (Fig. 4B) [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF][START_REF] Chudakov | Chromophore environment provides clue to 'kindling fluorescent protein' riddle[END_REF].

Hence the asFP595-Ser157Val mutant seems to be reminiscent of the situation observed in Dronpa on .

rsFastLime: A fast switching RSFP

In their fluorescent-states, the chromophores of both Dronpa on and on-state asFP595 adopt a cis isomerization state. Given the overall similarities of the chromophore environments of both proteins it is tempting to assume that off-state Dronpa, like off-state asFP595, adopts a trans conformation.

As described above, in Dronpa on Val157 seems to sterically oppose a trans conformation of the chromophore (Fig. 5A). We reasoned that removal of the valine side chain would be more compatible with a trans chromophore and consequently might reduce the energy barrier for photochromic switching in Dronpa. To test this hypothesis, we generated the Dronpa variant Val157Gly. In line with the prediction, the switching speed of Dronpa-Val157Gly is increased (Fig. 5B). Under the employed light intensities the blue light induced switch-off half-time t 1, Suppl. Fig. 3). The quantum yield ( FL ) of Dronpa is 0.85 [START_REF] Habuchi | Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa[END_REF], whereas Dronpa-Val157Gly exhibits a FL of 0.77 (Tab. 1). This accounts for the only slightly reduced overall fluorescence intensity of Dronpa-Val157Gly when the protein is expressed in cells (Fig. 5B). Because of the favorable combinations of properties (namely fast switching kinetics and high quantum yield), which render

DISCUSSION

In the present work we have determined a high-resolution equilibrium state structure of the RSFP Dronpa. In the fluorescent equilibrium state the CYG chromophore adopts a cis conformation. Relying on structural comparisons to another RSFP, namely asFP595 and its variants, we predict that a cis-trans isomerization may also accompany the switching-off of Dronpa. Based on this prediction we have generated two fast switching variants, in which the switch off half-time t 1/2 off is reduced by a factor of ~ 50 (rsFastLime) or by more than 1000 (Dronpa-Met159Thr) compared to the original Dronpa .

Flexible anchoring of the chromophore is required for reversible switching

The chromophore environment of Dronpa on exhibits a striking structural similarity to that of KikG, a non-switchable fluorescent GFP-like protein from the stony coral Favia favus (PDM 1XSS; [START_REF] Tsutsui | Semirational engineering of a coral fluorescent protein into an efficient highlighter[END_REF]) All residues in the direct neighborhoods of the two chromophores are identical in Dronpa on and KikG (Fig. 4C,D). The single major structural difference within the immediate chromophore environments are the chromophores themselves. In contrast to the CYG chromophore of Dronpa, the KikG chromophore is formed from an Asp62-Tyr63-Gly64 tripeptide. In KikG, the carboxyl group of Asp62 maintains four water mediated hydrogen bonds to Gln38, Phe61, Tyr116 and Leu209, thus firmly stabilizing this part of the chromophore, likely inhibiting considerable relocations of the chromophore. Comparable linkages are missing in Dronpa on , since the thiol group of Cys62 does not engage in any hydrogen bonds. Rather, this portion of the CYG chromphore is primarily anchored by van-der-Waals interactions, which may allow movement of the chromophore within the protein matrix.

In line with this interpretation, molecular dynamics calculations on asFP595 demonstrated that structural flexibility of the chromophore facilitates photochromism [START_REF] Andresen | Structure and mechanism of the reversible photoswitch of a fluorescent protein[END_REF]. In asFP595, residue 62 is a methionine that is not capable of forming a stabilizing hydrogen bond, similar to Cys62 in Dronpa on . Furthermore, in asFP595 flexibility is further increased by a main chain break between Cys61 and the MYG. This increased flexibility appears to further reduce the energy barrier for a cis-trans isomerization and thus for photoswitching, which may be reflected in the lowered stability of the on-state asFP595. After photoswitching the fluorescent state of asFP595 has a half life of only ~7 s before it reverts back into the non-fluorescent equilibrium state [START_REF] Chudakov | Chromophore environment provides clue to 'kindling fluorescent protein' riddle[END_REF], whereas Dronpa is almost bistable, with the non-fluorescent state exhibiting a half-life of ~14 hours (Table 1, Suppl. Fig. 3). In summary, the ability to make a flexible adjustment of the chromophore position with respect to the protein matrix appears to be essential for efficient reversible switching.

Interplay of factors for reversible switching

We propose that several preconditions have to be met before efficient reversible photoswitching may occur in a fluorescent protein: i) The chromophore should exhibit sufficient flexibility to allow a light-driven cis-trans isomerization. ii) The chromophore environment must provide sufficient space to allow the chromophore to accommodate both isomerisation states. And iii) The chromophore environments in the cis and the trans position should be sufficiently distinct to influence the fluorescence properties of the chromophore. In Dronpa, the chromophore exists in an equilibrium between protonated non-fluorescent and deprotonated fluorescent forms [START_REF] Ando | Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting[END_REF][START_REF] Habuchi | Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa[END_REF].

Therefore, while a cis-trans isomerization appears to be a key event that accompanies photochromic switching in RSFPs, chromophore isomerization itself is not sufficient to fully explain the phenomenon of photochromism in RSFPs. Currently it is difficult to predict whether a specific isomerization state is fluorescent or not. It will be enlightening to understand the detailed interplay of chromophore planarity, environment and protonation state, which are all crucial for fluorescence, with the cis-trans isomerization of the chromophore.

Fast switchable protein rsFastLime

Modeling of a trans chromophore into the Dronpa on structure immediately revealed several spatial constraints, primarily imposed by the side chains of Arg66 and Val157. If there is a photoinduced cis-trans isomerization in Dronpa, it requires the movement of Arg66 and Val157, which in turn might induce spatial rearrangements of other residues. Therefore, the structural changes taking place in Dronpa upon photoswitching might be more complex than previously reported for asFP595. Still, the fact that exchange of either Val157 or Met159 by smaller residues accelerates switching supports the view that a cis-trans isomerization of the chromophore is a key event in photochromic switching of Dronpa.

Irrespective of the exact details of the mechanism, the outstanding properties of rsFastLime, namely its high quantum yield and its moderately accelerated switching behavior, render it a very promising protein for several applications, in particular for breaking the diffraction barrier in far field fluorescence microscopy. For example, for RESOLFT super-resolution microscopy techniques using switchable fluorescent proteins, fast switching kinetics are mandatory [START_REF] Hell | Imaging and writing at the nanoscale with focused visible light through saturable optical transitions[END_REF][START_REF] Hell | Concepts for nanoscale resolution in fluorescence microscopy[END_REF].

The same applies for the recently proposed methods using individual molecule switching [START_REF] Betzig | Imaging intracellular fluorescent proteins at nanometer resolution[END_REF][START_REF] Rust | Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)[END_REF].

In Dronpa and its variants the wavelengths required for probing fluorescence is also inducing the on-to-off state conversion. For protein tracking applications in live cells, this property may prove to be a constraint for some applications, whereas it may be a virtue for others. The speed of this transition is expedited in rsFastLime. Hence for long term protein tracking approaches which do not require a fast switching behavior the almost bistable Dronpa is likely better suited, whereas for faster movements requiring fast switching, rsFastLime might be advantageous.

.

Fig. 1 .Fig. 2 .

 12 Fig. 1. A) Dronpa on fluorescence emission spectra (excitation at 488 nm). Displayed is the normalized emission spectrum of Dronpa on in solution (dotted line) and of Dronpa on protein crystals (solid line). B) Overall structure of Dronpa on . The chromophore is shown in ball-andstick. N-and C-termini and secondary structure elements are labeled. All structure figures were prepared with PyMOL (http://pymol.sourceforge.net/). C) Amino acid sequence alignment of Dronpa, KikG and asFP595. Amino acid numbering according to Dronpa. The residues forming the chromophore are highlighted by bold letters. Identical residues are shaded in dark grey, positions at which two of the proteins share identical amino acids are shaded in light grey. Distinct amino acid residues in the immediate chromophore environments of Dronpa and asFP595 are shaded in black.

Fig. 3 .

 3 Fig. 3. Opening in the -barrel. A) Surface view of Dronpa on displaying an apparent opening in the -barrel connecting the p-hydroxyphenyl moiety of the chromophore (in ball-and-stick; colorcoded by atom type as before) with the bulk solvent. Compared to Fig. 1C, the molecule has been rotated 25 ° about the horizontal axis (top to back). The cleft on the protein surface is highlighted by a dashed line. The inset shows a blow-up of the opening filled with three well-ordered water molecules. B) Partial sequence alignment of various fluorescent proteins. The sequence Trp-Glu-

Fig. 4 .

 4 Fig. 4. Comparison of Dronpa on with asFP595 variants and KikG. A) Comparison of the chromophore environment of Dronpa on with on-state asFP595-A142S (PDB ID 2A56; [7]) after global superimposition of the proteins. Backbone tube and carbon atoms (except chromophore) of Dronpa on are in gray, those of asFP595 are in black. Selected residues are labeled in gray. asFP595 residues distinct from Dronpa are indicated in black. Carbon atoms of the Dronpa on (CYG) and the asFP595 (MYG) chromophores are in light and dark green, respectively. Otherwise, color coding as before. The view is identical to that of Fig. 2A (left side). B) Similar comparison of Dronpa on with asFP595-S157V (PDB ID 2A52; [7]) after global superimposition of the proteins. C) Comparison of the chromophore environments of Dronpa on (protein/CYGchromophore gray/light green) with KikG (PDB ID 1XSS; protein/DYG-chromophore black/dark green) after global superimposition of the proteins. All residues in the direct vicinity of the chromophores are identical and are shown in ball-and-stick. Water molecules are omitted for clarity. The only significant structural discrepancy is due to the first position in the respective chromophores, as highlighted by the rectangle. D) Representation of the boxed region in (C). Some residues are omitted for clarity. Hydrogen bonds are indicated by dashed lines. Water molecules are indicated as cyan (Dronpa on ) and blue (KikG) spheres.

Fig. 5 .

 5 Fig. 5. The fast switchable proteins rsFastLime and Dronpa-M159T. A) Representation of a hypothetical trans chromophore modelled into the Dronpa on structure. Without major adjustments in the protein backbone or side chain conformations, the side chains of R66 and V157 would sterically interfere with the chromophore in trans as indicated by the dotted van-der-Waals spheres. The views are identical to Fig. 2A. B) rsFastLime and Dronpa-M159T exhibit

Table 1

 1

	1/2	off is

Biochemical Journal Immediate Publication. Published on 22 Nov 2006 as manuscript BJ20061401 Copyright 2006 Biochemical Society

ACKNOWLEDGEMENTS

We thank Reinhard Lührmann for access to his infrastructure. We gratefully acknowledge Atsushi Miyawaki for providing us with the plasmid pRSETb-Dronpa. We thank Sylvia Löbermann for excellent technical assistance and Chaitanya Ullal for carefully reading the manuscript.

ABBREVIATIONS

RSFP, reversibly switchable fluorescent proteins; GFP, green fluorescent protein; RESOLFT, reversible saturable optical fluorescence transitions