Justyna Stec-Niemczyk

Katarzyna Pustelny

Magdalena Kisielewska

Michal Bista

Kevin T Boulware

Henning R Stennicke

Ida B Thogersen

Patrick S Daugherty

Jan J Enghild

Krzysztof Baczynski

Grzegorz M Popowicz

Adam Dubin
email: gdubin@mol.uj.edu.pl

Jan Potempa

Grzegorz Dubin
email: grzegorzdubin@hotmail.com

Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

Keywords: serine protease, structure, substrate specificity, Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen which antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Serine protease like proteins (Spl) encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins likely involved in virulence. Here we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolyzed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The likely physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel, significantly different mechanistic details.

Introduction

Species of Staphylococcus genus are commensal inhabitants of man and warm-blooded animal epithelium [START_REF] Lowy | Staphylococcus aureus infections[END_REF]. Nearly 30% of human population is persistently colonized by Staphylococcus aureus without visible adverse effects. At the same time the bacterium is a dangerous opportunistic pathogen. Microinjurys of skin or surgical wounds can lead to invasion of subepithelial tissues. S. aureus reveals a great ability to overcome host defense mechanisms and colonize diverse organs. The pathogen is equipped with a number of secreted factors facilitating superficial and systemic infections. Examples of soft tissue superficial infections include impetigo, folliculitis, abscesses, boils or infected lacerations [START_REF] Noble | Skin bacteriology and the role of Staphylococcus aureus in infection[END_REF]. Deep systemic infections are more dangerous and often life-threatening (e.g. endocarditis, meningitis, septic arthritis, pneumonia or osteomyelitis) [START_REF] Archer | Staphylococcus aureus: a well-armed pathogen[END_REF]. The most serious outcome of S. aureus infection is septicemia. Moreover, the bacterium is responsible for several toxinoses, such as staphylococcal scalded skin syndrome (SSSS), toxic shock syndrome (TSS) and food poisoning. The wide variety and severity of infections caused by S. aureus together with a frightening increase of multi-antibiotic resistance and the lack of effective vaccines poses an urgent need for novel therapeutic strategies [START_REF] Maskalyk | Antimicrobial resistance takes another step forward[END_REF][START_REF] Shorr | Epidemiology of staphylococcal resistance[END_REF]. This in turn necessitates deeper understanding of staphylococcal physiology and pathogenicity pathways.

Virulence factors responsible for staphylococcal pathogenicity encompass secreted toxins, immune-modulatory and adhesion molecules, signaling factors and extracellular enzymes, including a number of proteases [START_REF] Lindsay | Staphylococcus aureus: superbug, super genome?[END_REF]. The role of cysteine proteases staphopain A and B, metalloprotease aureolysin, serine protease V8 (glutamylendopeptidase) and epidermolytic toxins in staphylococcal virulence is documented in multiple reports [START_REF] Dubin | Extracellular proteases of Staphylococcus spp[END_REF][START_REF] Redpath | The role of the serine protease active site in the mode of action of epidermolytic toxin of Staphylococcus aureus[END_REF][START_REF] Shaw | Cytoplasmic control of premature activation of a secreted protease zymogen: deletion of staphostatin B (SspC) in Staphylococcus aureus 8325-4 yields a profound pleiotropic phenotype[END_REF][START_REF] Bailey | The epidermolytic (exfoliative) toxins of Staphylococcus aureus[END_REF]. Furthermore, screening of a library of staphylococcal proteins with antisera from patients with S. aureus endocarditis clearly showed that a new serine protease like protein, SplC, is expressed during infection [11]. Follow up investigations demonstrated that SplC is a product of a gene belonging to an operon encoding up to six homologous Spl proteases in various strains of S. aureus [START_REF] Reed | Molecular characterization of a novel Staphylococcus aureus serine protease operon[END_REF]. Despite their suggested involvement in pathogenesis, the Spl proteases remain poorly characterized. Similar to other staphylococcal exo-proteins, in vitro the spl operon is transcribed during early stationary phase of bacteria growth [START_REF] Ziebandt | The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus[END_REF]. It is postulated that similar regulation operates in vivo and is essential for dissemination of the pathogen from initial colonization sites. Therefore, Spl proteases together with multiple similarly regulated documented virulence factors may play a role in the second, invasive stage of the infection. The likely contribution of Spl proteases to S. aureus pathogenicity is further substantiated by structural similarities to established virulence factors, such as V8 protease and epidermolytic toxins, and clustering of the spl genes on a stable pathogenicity island (vSaβ) [14,[START_REF] Popowicz | Functional and structural characterization of Spl proteases from Staphylococcus aureus[END_REF].

To date out of six Spl proteins only SplB and SplC were studied in some details. The lack of proteolytic activity of SplC was explained on the atomic level by showing that the catalytic machinery is significantly distorted in the crystal structure of the protein. Moreover, crystal structure of highly specific protease SplB shown lack of an important element of serine-protease catalytic machinery, the oxyanion hole. By comparison to epidermolytic toxins authors speculated that binding of an appropriate substrate could induce activating changes in SplC, similarly to SplB which activity seems induced by binding of a substrate encompassing the sequence Trp-Glu-Leu-Gln [START_REF] Popowicz | Functional and structural characterization of Spl proteases from Staphylococcus aureus[END_REF]16].

In this report, to cast more light on staphylococcal proteases of spl operon we have produced SplA using heterologous expression system in B. subtilis and performed biochemical and structural characterization of the recombinant protein. We demonstrate a crucial effect of proper N-terminal trimming for SplA activity. Of interest, we characterize the enzyme strict specificity imposed by recognition of amino acid residues at P1, P2 and P3 positions of the substrate (nomenclature according to Schechter and Berger [17]). The structural basis of substrate preference and significance of the N-terminal residue of the mature protein in SplA activity are visualized at the atomic level and contrasted with type proteases of chymotrypsin family.

Experimental Bacterial strains, plasmids, protein expression and purification

Escherichia coli strains DH5α and BL21(DE3) (Invitrogen) were used respectively for genetic manipulations and protein expression. For production of GST-GS-SplA (GS indicates that following tag removal, a Gly-Ser artifact remains at the N-terminus of SplA compared to the native protein), the splA gene (without a signal peptide) was PCR amplified from the genomic DNA of S. aureus strain 8325-4 and cloned into BamHI/XhoI sites of pGEX-5T [18]. GST-YLYS-Staphostatin A fusion protein (GST-YLYS-ScpB; YLYS indicates a Tyr-Leu-Tyr-Ser sequence engineered between two globular fusion partners) was produced as previously described [19] with the exception that in the middle part of the construct the amino acid sequence recognized by thrombin (LVPRGS) was exchanged by site directed mutagenesis into consensus amino acid sequence recognized by SplA (LVYLYS). For SplA production B. subtilis strain WB800 carrying a pWB980 vector [START_REF] Wu | Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis[END_REF] with the splA gene (without a native signal peptide) inserted at HindIII/XhoI sites was used.

E. coli cells containing pGEX5T-GS-SplA vector were cultured at 37˚C in LB containing ampicillin (100 µg/mL) until OD 600nm reached 0.8 absorbance units and latter at room temperature. Protein expression was induced with 1mM IPTG when the cultures reached OD 600nm of 1.2 absorbance units. After 3h cells were collected in PBS and lysed by sonication. A protein of interest was recovered using Glutathione-Sepharose (Amersham Biosciences). GST fusion tag was removed using thrombin (BioCentrum, Krakow). GS-SplA was further purified by ion exchange chromatography (SoruceS; Amersham Biosciences; 50mM Sodium Acetate pH 6.5) followed by gel filtration on Superdex 75pg (Amersham Biosciences) equilibrated with PBS. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Routinely recombinant SplA was produced in B. subtilis WB800 carrying pWB980-SplA plasmid driving a constitutive secretory expression. The cells were cultured at 37˚C overnight in TSB medium containing kanamycin (10 µg/mL). Cell free supernatants were collected by centrifugation and proteins were precipitated with ammonium sulfate at 80% saturation at 4˚C (561 g/L). Precipitated proteins were collected by centrifugation, resuspended in buffer A (50 mM amonium acetate, pH 5.5) and dialyzed overnight against the same buffer. SplA was recovered by ion exchange chromatography on SP-Sepharose Fast Flow (Amersham Biosciences) equilibrated with buffer A. Fractions with conductivity between 27 mS/cm and 37 mS/cm, containing proteolytic activity were pulled, and rechromatographed on SourceS (Amersham Biosciences) in buffer B (50 mM amonium acetate, pH 5.0). Proteolytically active fractions were pulled and further purified on Superdex 75pg (Amersham Biosciences) equilibrated with crystallization buffer (5 mM Tris, 50 mM NaCl, pH 8.0).

GST-YLYS-ScpB was purified as previously described for GST-ScpB [19].

Proteolytic activity assays

Activity of SplA was routinely tested by casein zymography [21]. Briefly, samples were incubated with SDS loading buffer (without reducing agent) in 37˚C for ½ hour and separated on 12% (w/v) polyacrylamide gel containing 0.1% β-casein.

Following electrophoresis the gel was washed for 30 min in 2.5% (v/v) Triton X-100 and developed for 3 hours or overnight in 100 mM Tris (pH 7.8), 100 mM NaCl, 5 mM EDTA at 37˚C. Zones of proteolysis were visualized by staining with Amido Black.

Synthetic chromogenic substrates tested included: H-AF-pNa, H-GF-pNa, N-Suc-F-pNa, N-Suc-AAPF-pNa, N-Ac-LY-pNa, N-Suc-LF-pNa and N-Suc-AAA-pNa. SplA (20 µg) was incubated with 0.5 mM substrate in a total volume of 200 µL 100 mM Tris-HCl, pH 7.0 for 16h at 37°C in a microtitration plate. Following incubation OD at 405 nm was measured using a plate reader. pH optimum of SplA was determined using N-Suc-AAPF-pNa. This substrate was also used to assay the effect of synthetic serine protease inhibitors, including phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and 3,4-Dichloroisocumarin (DCI) on SplA activity.

A number of native proteins and peptides including: chicken egg white lysozyme and ovalbumin, soybean trypsin inhibitor, human serum transferrin, bovine serum albumin, RNAse, cytochrome c, β-casein, galanine, goat IgGs, bovine and human fibrinogen and whale mioglobin were tested for hydrolysis by SplA in Bis-Tris buffer (pH 6.5). Moreover, proteins denatured by chemical modification, including: carboxymethylated chicken egg white lysozyme and bovine pancreatic trypsin inhibitor BPTI, as well as apomioglobin and apocytochrome c were evaluated as substrates for SplA.

Human serum serine protease inhibitors including α-1-antitrypsin, α-1antichymotrypsin, antithrombin and α 2 -macroglobulin were tested for inhibition of SplA activity. The residual activity was determined using GST-YLYS-ScpB as a substrate after 30min preincubation of SplA with a tested inhibitor at 1:100 molar ratio. In addition, SplAinhibitor reaction mixtures after 15 minutes of incubation at 37˚C were analyzed by SDS-PAGE for inhibitory complex formation or potential inhibitor cleavage.

Edman degradation and MALDI-TOF MS analysis

For N-terminal sequence determination, protein cleavage products were separated by SDS-PAGE, electro-blotted on PVDF and sequenced by automated Edman degradation sequencing at BioCentrum (Krakow, Poland).

For determination of molecular weights the protein cleavage products were separated by HPLC and subjected to MALDI-TOF MS (matrix-assisted laser desorption/ionizationtime-of-flight) analysis. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Positional scanning substrate libraries

To assess the preference of SplA for amino acid residue at P1 substrate position positional scanning substrate libraries were used as described previously [22].

Cellular library of peptide substrates (CLiPS)

A complete consensus sequence recognized and cleaved by SplA protease was discerned using a CLiPS methodology as previously described [START_REF] Boulware | Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)[END_REF]. In brief, a library of 6x10 7 clones displaying a bait polypeptide containing an SH3 domain binding motif, five consecutive randomized amino acids linker and a streptavidin binding peptide ligand was screened for proper display and SplA hydrolysis using fluorescence activated cell sorting (FACS). Clones with intact baits show red and green fluorescence after incubation with phycoerythrin conjugated streptavidin (50 nM) and SH3 domain conjugated Green Fluorescent Protein GFP (250 nM). Clones displaying substrates that were specifically hydrolyzed after 16 hours of incubation with 0.5 µM SplA were obtained by sorting the green cells only. After three rounds of sorting individual clones were analyzed for hydrolysis by incubating with 0.5 µM SplA for 3 hours, and clones demonstrating hydrolysis were sequenced (Table I).

Protein crystallization and structure solution

Purified active SplA and SplA reacted with phosphonate inhibitor [START_REF] Oleksyszyn | Irreversible inhibition of serine proteases by peptidyl derivatives of alpha-aminoalkylphosphonate diphenyl esters[END_REF]25] were concentrated by ultrafiltration to ~30 mg/ml in crystallization buffer and used for initial screening. After few weeks crystals appeared under several different conditions. For active SplA following further optimization a best single monocrystal of about 0.3 mm length was obtained using Hampton Research Index 23 (2.1 M DL-Malic acid, pH 7.0) and was used for measurements. The crystal was mounted in a glass capillary and the diffraction data were collected on a rotating anode laboratory source at room temperature. Best single monocrystal of inhibited SplA obtained from an in-house screen (0.1M Hepes pH 7.5, 0.2M (NH 4 ) 2 SO 4 , 30% PEG 6000) [START_REF] Jancarik | Sparse matrix sampling: a screening method for crystallization of proteins[END_REF] was cryo-preserved in liquid nitrogen. The diffraction data were collected at SLS beamline X10SA.

Data were indexed and integrated using MOSFLM [START_REF] Leslie | The integration of macromolecular diffraction data[END_REF]28]. Following computational steps were done using programs contained in CCP4 package [29]. The data were scaled in SCALA [30,31]. Molecular replacement solution was found using Phaser [START_REF] Mccoy | Solving structures of protein complexes by molecular replacement with Phaser[END_REF] with SplC (PDB ID 2as9) as a search model. SplA model was manually built in resulting electron density maps. Restrained refinement of bond lengths and angles was imposed with Refmac 5.0 [33]. Throughout the refinement 5% of the reflections were used for cross-validation analysis [START_REF] Brunger | Free R value: a novel statistical quantity for assessing the accuracy of crystal structures[END_REF], and the behavior of R free was employed to monitor the refinement strategy. Water molecules were added using Arp/Warp [START_REF] Lamzin | Automated refinement of protein models[END_REF] and subsequently manually inspected. All detailed features described in the text were validated using simulated annealing omit maps computed with CNS [36,[START_REF] Brunger | Crystallography & NMR system: A new software suite for macromolecular structure determination[END_REF]. The final models were deposited at Protein Data Bank under accession numbers: 2w7u and 2w7s. The data collection and refinement statistics are summarized in Table II.

Homology modeling

φ and ψ mainchain angles of a substrate were modeled as found in canonical inhibitors of serine proteases which bind in a manner resembling natural substrates [38]. Substrate binding surface of SplA was identified based on homology to S1 family proteases. The substrate model was manually docked into the substrate binding surface of the enzyme. Minor corrections of the mainchain disposition within the template derived from canonical inhibitors were performed to optimize recognized hydrogen bonds [39]. The side chains were added according to the identified consensus sequence recognized by SplA. Sidechain disposition was modeled based on structures of S1 family proteases complexed with synthetic and protein inhibitors. Following hydrogen atoms were added to the model using AddH module of UCSF Chimera package [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF], potentials were assigned according to Amber 1994 force field [START_REF] Cornell | A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules[END_REF] and the energy of the complex was minimized using MMTK module of Chimera with default values [START_REF] Hinsen | The molecular modeling toolkit: A new approach to molecular simulations[END_REF]. Manual structure edition, structure analysis and molecular graphics were done using modules of Chimera and spdbv.

Bioinformatic analysis

Human (Homo sapiens, taxid: 9606) and staphylococcal (S. aureus, taxid:1280) proteomes were analyzed for potential cleavage sites with the deduced consensus sequence recognized by SplA (YLYS) using BLAST algroithm and Uniprot data base. Initial hits were manually examined to remove redundant sequences. The remaining proteins were analyzed for tissue and cellular localization (for a list of all nonredundant hits see supplementary materials table S1) to pinpoint those directly spacially colocalized with staphylococci during commensal colonization and/or infection.

Results

Production of recombinant SplA

Initially SplA was expressed as a GST fusion protein in E. coli and the tag was cleaved off with thrombin during purification procedure. Thus produced recombinant GS-SplA was inactive in zymographic assays using gelatin and β-casein as substrates. As was the case with related protease SplB [START_REF] Popowicz | Functional and structural characterization of Spl proteases from Staphylococcus aureus[END_REF]16] the lack of the proteolytic activity could have been related to the presence of a Gly-Ser dipeptide, a remnant of the thrombin cleavage site, on the N-terminus of GS-SplA. To investigate this option, recombinant SplA was directed onto secretory pathway to ensure proper N-terminal trimming by signal peptidase. Bacillus subtilis expression plasmid pWB980 driving constitutive production and secretion of SplA was constructed and transformed into a knockout strain WB800 lacking majority of endogenous extracellular proteases. Indeed, obtained in this way, the recombinant SplA showed proteolytic activity in zymography indicating the importance of N-terminus for enzymatic activity. The system yielded around 10 mg purified protein per 1 liter of a starting culture. Thus produced protein was used in all further analyses.

Substrate specificity of SplA

To characterize SplA activity we have evaluated an array of synthetic p-nitroanilide substrates. Among tested compounds (see experimental procedures) only N-Suc-Ala-Ala-Pro-Phe-pNa was hydrolyzed, though very inefficiently. Nevertheless, using this substrate we estimated the pH optimum of the protease at pH 6.5 (Bis-Tris buffer). Moreover, we have demonstrated that amongst common low molecular weight serine protease inhibitors tested only 3,4-dichloroisocoumarin inhibited SplA.

The P1 substrate preference of SplA was determined by screening the enzyme activity against a panel of combinatorial libraries of synthetic fluorescent substrates with a general structure of Ac-Xaa-Xaa-Xaa-P1-aminomethylcoumarin. SplA exclusively hydrolyzed substrates with monocyclic aromatic residues in P1, namely tyrosine and to a lower extent phenylalanine (Figure 1.) The substrate specificity was further evaluated using MS and Nterminal microsequencing analysis of cleavage products of β-casein and carboxymethylated chicken lysozyme. The results confirmed SplA preference for phenylalanine and tyrosine in P1 (Table I). The cleavage kinetics of casein and lysozyme was however extremely slow requiring an excess of an enzyme and prolonged incubation times. Significantly, none of other tested native and denatured protein substrates or human serum serine protease inhibitors (for complete list see experimental procedures), all containing multiple Phe and Tyr residues were cleaved by SplA. The above results collectively suggest that SplA specificity is determined not only in P1, but also in further subsites.

To determine the preference of SplA for particular residues in P(n) as well as P(n)' positions and to account for possible subsites cooperation we have applied a high-throughput CLiPS methodology [START_REF] Boulware | Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)[END_REF]. A library presenting five consecutive randomized amino acids in a context of a cell surface protein was selected for SplA cleavage. The analysis demonstrated that SplA recognizes and cleaves a consensus sequence of four consecutive residues Trp/Tyr-Leu-Tyr-Thr/Ser (Table I).

Taking into account the P1 preference for monocyclic aromatic residues there are two possible cleavage sites in the consensus sequence. To determine which peptide bond is hydrolyzed and demonstrate that SplA can efficiently cleave proteins containing this sequential motif we have constructed a fusion protein of two globular partners with a linker containing the identified consensus amino acid sequence (YLYS). At 1:100 enzyme to substrate molar ratio, 160 µg of purified fusion protein (GST-YLYS-ScpB) was completely and specifically split into free GST and ScpB in 15 minutes (supplementary materials -Figure S2). Edman degradation sequencing of the released N-terminus revealed hydrolysis of the Tyr-Ser peptide bond within the consensus sequence.

Collectively, these data demonstrates that SplA is a highly selective protease which can cleave efficiently only substrates containing the Trp/Tyr-Leu-Tyr-Thr/Ser sequence motif.

Overall crystal structure of SplA protease

To explain narrow specificity of SplA at the atomic level we have crystallized and solved the enzyme structure. Two independently determined structures are discussed (Table II). A 2.43Å structure of an active SplA protease and 1.8Å structure of a protease reacted with phosphonate inhibitor. Both structures are essentially identical (the average RMSD of backbone atoms for all eight models equals 0.45) since the inhibitor has hydrolized during crystallization and is not accounted for by electron density. Therefore, the low resolution model is discussed mainly to demonstrate said identity, importance of which is stressed in the discussion. Unless otherwise indicated 1.8 Å structure is used to discuss the structural details.

The protein shows a chymotrypsin-like fold. SplA molecule consists of two domains (I and II), each of which is built of six anti-parallel β-strands folded into a β-barrel (Figure 2). The domains are connected via a stretched linker containing amino acids Asn95 through Val109. Domain I, although composed mostly of N-terminal residues, (Tyr15-Phe94) also contains a C-terminal fragment (Thr187-Lys200) encompassing an α-helix (Pro188-Asn197). Domain II is build of residues Lys110-Phe186 and the most N-terminal portion of the protein (Glu1-Ile7). The active site of the enzyme is located at the interface of the two barrels and consists of residues His39, Asp78 and Ser154 conserved in all enzymatically active chymotrypsin-like proteases.

The asymmetric unit contains four molecules. Superimposition of Cα atoms of those molecules yields average RMSD of 0.5 Å. Small differences are visible in positioning of surface loop Lys58-Gly63 which is characterized by some flexibility as evidenced by high B factors and week electron density, and in regions Thr87-Gly91 and Ala104-Ala107 which are affected by crystal packing interactions. However, most pronounced differences due to crystal packing are found in the region His39-Ser52. Residues Asn37 through Ile46 form a helix in models A and B which protrudes with its C-terminal part away the globular core of the molecule. In models C and D respectively residues 42-47 and 42-43 are not defined by electron density. The average B factors for residues 38-49 are higher than of surrounding residues. In all models the entire discussed region forms close contacts with surrounding molecules. Of note on top of the protrusion the hydrophobic side chain of Phe47 is buried in a shallow pocket of adjacent molecule.

Primary sequence analysis shows significant homology between SplA and other chymotrypsin-like serine proteases from S. aureus (38% with V8 protease and 27% with epidermolytic toxins ETA and ETB). Accordingly, the superimposition of the Cα trace of SplA with that of V8 protease yields RMSD of 1.0 Å (over 165 Cα atoms) and with that of epidermolytic toxins A and B of 1.1 Å (153 Cα atoms) and 1.1 Å (146 Cα atoms), respectively.

Catalytic Triad

The conformational restraints of catalytic triad, an essential element of catalytic machinery of S1 family serine proteases, are not satisfied in the structure of SplA. Sequence alignment and superposition of V8 protease, SplB and SplA models demonstrates the presence of the three essential residues (His39, Asp78 and Ser154 in SplA), nevertheless the crucial hydrogen bonding network between the sidechains of those residues is not present in SplA structure (Figure 3). Although, when whole molecules are superposed, the positions of Asp78 and Ser154 of SplA are similar to those of correspondig residues in V8 and SplB, His39 sidechain is differently positioned. The distances between N ε of His39 and O γ of Ser154 and between N δ of His39 and O δ of Asp78 are significantly longer. Such distances indicate only week or no hydrogen bonding as opposed to strong hydrogen bonding observed in canonical structures [43]. In its extreme in model B of 2.43 angstrom structure the N ε of His39 is hydrogen bonded to a carbonyl oxygen of A122 of adjacent molecule and in model A of the same structure a water molecule is hydrogen bonded between N δ of His39 and O δ of Asp78. The reason for such atypical His 39 conformation as compared to type proteases of family S1 is evident from what was discussed in previous section. The position of 37-46 helix is affected by crystal packing and so is the placement of H39 imidazole ring, though His39 is not directly involved in intermolecular contacts (besides His39_B of 2.43 angstrom structure). Described unusual arrangement of catalytic triad histidine is not compatible with the standard mechanism of catalysis. However, since model B of 1.8 angstrom structure shows almost canonical hydrogen bonding and we unambigously demonstrated here that SplA is catalytically active against peptide substrates we conclude that the discussed phenomenon is an effect of crystal packing interactions.

Oxyanion hole

In S1 family of serine proteases the oxyanion hole stabilizes a tetrahedral transition-state intermediate of the carbonyl carbon atom of a scissile peptide bond by compensating the negative charge formed on the carbonyl oxygen. In contrast to what was previously reported for closely related enzymes: SplB protease [16] and ETs [44] the conformation and Pro151-Gly152 peptide bond in SplA supports a functional oxyanion hole similarly to previously reported structure of V8 protease. In SplA Pro151 has average main chain angles of φ = -44º and ψ = 134º, which are similar to those of corresponding Gly166 in V8 protease (φ = -50º and ψ = 130º) and different from those of corresponding Pro192 in ETA where φ = -48º and ψ = -40º and corresponding Ser154 in SplB (φ = -62º and ψ = -34º). The Gly 152 has main chain angles of φ = 110º and ψ = -26º, which are again similar to those of corresponding Gly167 in V8 protease (φ = 121º and ψ = -30º) and different from those of corresponding Gly193 in ETA where φ = -60º and ψ = -22º and corresponding Gly155 in SplB (φ = -63º and ψ = -17º). In all mentioned enzymes φ and ψ angles of following and proceeding residues correspond to those found in type S1 family proteases. The described conformation of Pro151-Gly152 peptide bond in SplA is stabilized by hydrogen bonding of carbonyl oxygen of Pro151 with the amide of Pro119-Lys120 peptide bond. Since inhibitor treatment might have resulted in the observed placement of Pro151-Gly152 peptide bond the 2.43 Å structure was carefully examined in the region in question. No evidence was found to support any alternative placement of the carbonyl oxygen of the described bond (for details see supporting information). Therefore, apart from the described distortion of catalytic triad histidine residue the catalytic machinery of SplA corresponds to that found in type proteases of S1 family.

Specificity pockets

Substrate specificity of family S1 proteases is commonly determined by pronounced S1-P1 interactions. Accordingly, the S1 pocket of SplA is an easily distinguishable, spacious, bowl-shaped cavity. It is delineated by loops I (Ala149-Ser154; [39]) and II (Gly174-Glu179) and strands Leu169-Ser173 and Lys180-Phe182. The cavity is lined with non-polar side chains of Ala149, Leu169 and Cβ atoms of Asn153 and Ser178 and mainchain atoms of mentioned secondary structures save for the strand Lys180-Phe182 which is buried and only stabilizes the pocket. As such it closely resembles the S1 pocket of chymotrypsin and should easily accommodate bulky side chains of Tyr and Phe as determined in specificity assays. Homology modeling of substrate docking based on multiple available structures of synthetic peptide and canonical protein inhibitors in complex with S1 family proteases demonstrates that the side chain oxygen of Asn181 which reaches the bottom of the S1 pocket in SplA can possibly interact through hydrogen bond with Tyr (P1) side chain hydroxyl of the substrate (Figure 4). The modeling also defines the S2 subsite. The mainchain of the substrate adopts an extended beta-sheet conformation and interacts via hydrogen bonds with antiparallel beta-strand of the enzyme formed by residues Leu169-Gly172. The branched side chain of leucine preferred in P2 docks into a shallow cleft formed by the side chains of two catalytic triad residues, His39 and Asp78, and further flanked by side chains of Ala171 and Tyr170. Contrary to observed strong preference for aromatic side chains at P3 substrate position the S3 subsite is not clearly defined. Comparisons with structures of serine proteases-inhibitor complexes and energy minimization of SplA -YLY peptide complex give unambiguous results. It therefore seems likely that an induced fit mechanism may be involved.

Hydrogen bond network at the N-terminal

Precise N-terminal trimming is a prerequisite for activity of type proteases of chymotrypsin family. Similarly, we demonstrate here that the addition of two aminoacids at the N-terminus of SplA renders the protein inactive. A likely explanation is provided by the crystal structure. The N-terminal glutamate it is firmly positioned on the surface of the molecule through a network of hydrogen bonds (Figure 5). The mainchain amide and carbonyl oxygen of Glu1 are hydrogen bonded respectively mainchain carbonyl oxygen of Asp146 and mainchain amide of Tyr148 as in a parallel β-sheet. The N-terminus amide is further hydrogen bonded to a sidechain of Glu179 while Glu 1 backbone carbonyl forms an additional hydrogen bond with a water molecule which is buried in the structure and further coordinated by the mainchain amide of Asn3. The side chain of Glu1 forms a salt bridge with the side chain of Arg112 and further hydrogen bonds with a water molecule and the sidechain of Thr132. Asn3 begins a terminal strand of an antiparallel β-sheet. The tightly positioned residues of Glu1 and Asn3 hold in place the Lys2 residue which by itself makes no pronounced contacts. Any N-terminal extention is not sterically compatible with the described descirbed Glu1 positioning and thus requires breaking of the hydrogen bond network described.

Putative in vivo SplA targets

A search of human and staphylococcal proteomes with SplA consensus cleavage sequence suggests likely in vivo targets of the enzyme. Out of 90 identified human and 3 staphylococcal proteins (supplementary materials, table S1), after analysis of tissue and cellular context together with the bacteria localization during commensal colonization as well as infection, two emerge as likely targets: mucin-16 (Q8WX52) and olfactory receptor 5I1 (Q13606).

Discussion

Similarly to SplB [START_REF] Popowicz | Functional and structural characterization of Spl proteases from Staphylococcus aureus[END_REF], the proper N-terminal processing is necessary for the enzymatic activity of SplA. In a closely related V8 protease of S1B family the N-terminal amino group released by cleavage of propeptide penetrates into the S1 pocket and it is directly responsible for charge neutralization of P1 glutamic acid of a scissile peptide bond. Such mechanism follows the classical pathway of S1A family serine protease zymogen activation, the most thoroughly studied examples of which include digestive tract proteases [START_REF] Khan | Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes[END_REF]. There, as in a multitude of other serine proteases the new N-terminus liberated upon proteolytic processing of a precursor is directly involved in a formation of a functional primary specificity pocket (Figure 5) [46][START_REF] Wang | Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution[END_REF][START_REF] Bode | Natural protein proteinase inhibitors and their interaction with proteinases[END_REF]. Sequence alignment of SplA and V8 protease demonstrates that the polypeptide chain of mature SplA is four amino-acids shorter at the N-terminus than that of V8 protease, making similar penetration in the former enzyme unlikely. Indeed, analysis of the crystal structure of SplA reveals a lack of direct involvement of the N-terminus in the formation of the S1 pocket. Nevertheless, the artificially, N-terminally elongated forms of Spl proteases, Gly-Ser-SplB [START_REF] Popowicz | Functional and structural characterization of Spl proteases from Staphylococcus aureus[END_REF] and Gly-Ser-SplA (this work) show no activity in zymography. The molecular mechanism by which the N-terminal extension controls activity of SplA protease is less obvious. The N-terminus is distant from either catalytic machinery or substrate recognition surface. Pronounced structural changes upon N-terminal elongation are unlikely since 1D NMR spectra of SplA and GS-SplA are almost identical (data not shown). Therefore, most probably only distinct conformational changes and possibly small rearangements in a network of hydrogen bonds regulate catalysis. Hydrogen bonding of Glu1 amide to a sidechain of Glu179, a residue close to P1 specificity pocket suggest influence on substrate recognition, however certainly the mechanism is distinct to that observed in type proteases of family S1. A structure of N-terminally elongated SplA (ex. GS-SplA) would probably help to clarify the mechanism though it may involve a substantial dynamic constituent which might be hard to sensibly model based on static crystallographic data. The inactivity of N-terminally elongated forms of Spl proteases seems not only a cloning artifact but rather a physiologically relevant phenomenon. Most proteolytic enzymes are syntesized as inactive zymogens as a protection against unwanted proteolysis. Only at target sites the propeptides are removed to release activity. Spl proteases lack propeptides of any kind and are secreted directly in an active form being obviously harmless for the bacterium while outside the cell. The latter is similarly true for staphopains (Staphylococcus arueus cysteine proteases) which are found in a mature, proteolytically active form in the culture media. However, for efficient production of extracellular staphopains specific intracellular inhibitors are necessary. It was demonstrated that knocking out these inhibitors decreases the viability of the bacterium. This demonstrates that a small amount of enzyme is misdirected into cytoplasm [START_REF] Shaw | Cytoplasmic control of premature activation of a secreted protease zymogen: deletion of staphostatin B (SspC) in Staphylococcus aureus 8325-4 yields a profound pleiotropic phenotype[END_REF]. One may suspect that similarly a small amount of every secretory protein and in particular of Spl proteases reamins inside the cell. This pool however contains a signal peptide at the N-terminus (which is cleaved off by a signal peptidase only upon secretion) and thus remains catalytically inactive. As such the signal peptide acts both as a secretion signal and a propeptide controlling the enzyme activity which likely explains the lack of demonstrated intracellular serine protease inhibitors in staphylococci.

The high substrate specificity of SplA protease, defined by three consecutive residues of a substrate, is uncommon among S1 family proteases. Homology modeling of substrate binding clearly defined the structural determinants of P1 and P2 preference. The strong propensity for aromatic P3 substrate residue could not have been satisfactory explained by modeling suggesting an induced fit mechanism might be involved. A crystal structure of SplA complexed with a substrate analogue is therfore desirable to clarify this issue. Parallel to the classical subsite based substrate recognition, oxyanion hole distortion was suggested to take part in specificity determination of SplB [16]. In SplA the arrangement of the Ser154 and Gly152 amides is similar to that observed in type serine proteases and different from that in SplB therefore, similar mechanism is not involved here.

Many proteases produced by pathogenic bacteria play crucial role in virulence. The broad substrate specificity enzymes are generally involved in unspecific processes like acquisition of nutrients and dissemination (both associated with tissue damage). In turn, highly specific proteases often act as dedicated toxins exerting activity on a narrow subset of proteins or even on a single substrate. The narrow specificity of SplA suggests a similar role. Deciphering the role of specific proteases in virulence is most interesting, but also a highly Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

H P F A V E P F T F L L Y Q Chicken carboxymethylated lysozyme S T D Y G Synthetic pNa substrate A A P F pNa CLiPS (Cellular Library of Peptide Substrates) * R W L Y (S) (S) W L Y S G W L Y (S) I Y E Y A L Y E Y (S) V Y E Y (S) V Y M Y (S) V Y A Y S F Y T Y S L Y L Y G A F L Y S T F L Y (S) I V L Y T V V L Y T Y W L S T Y W M N T Y W W Y T A W L Y (

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081351 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081351

Biochemical

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Biochemical

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Biochemical

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Biochemical

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  challenging undertaking. A search for physiological substrate of ETs took nearly 30 years to achieve[49]. Considering pronounced similarity of Spl proteases to staphylococcal virulence factors, location of the spl operon within the pathogenicity island [14], expression during staphylococcal endocarditis [11] and highly limited substrate specificity it is tempting to speculate that Spl proteases represent yet another group of S. aureus specific toxins. Analysis of human proteome with the deduced consensus sequence recognized by SplA points to two fascinating putative targets. Olfactory receptor (OR) 5I1 (Q13606), a member of a large family of transmembrane sensory proteins is clearly found in the primary colonization site of S. aureus. One may speculate that its cleavage may facilitate the colonization and/or persistence of S. aureus in the nares, or facilitate dissemination from this primary niche into the human body by yet unknown mechanisms. What is certain is that consensus cleavage site of SplB protease was found in another members of ORs family[16]. Another identified protein, mucin-16 (MUC16), possesses two putative cleavage sites for SplA. Mucin-16 is a heavily Oglycosylated transmembrane protein, which is expressed by the ocular surface epithelia in which it helps provide a disadhesive protection barrier[START_REF] Blalock | Functions of MUC16 in corneal epithelial cells[END_REF]. Staphylococcus aureus is a leading cause of bacterial keratitis in adults that can result in irreversible corneal scarring and loss of visual acuity. To date mainly Alpha-and Beta-toxin of S. aureus were convincingly demonstrated as virulence factors in corneal disease. In the context of staphylococcal corneal infection, cleavage of MUC16 by SplA is worth detailed investigation. Although the above findings on possible physiological role of SplA protease are highly speculative, they open an exciting avenue for future research on related aspects of staphylococcal physiology.

Figure 1 P1

 1 Figure 1 P1 substrate preference of SplA.Specificity of recombinant SplA was determined using a set of positional scanning libraries of general structure Ac-Xaa-Xaa-Xaa-P1-AMC as described in the Experimental section. Vertical axis represents the rate of AMC production as a percentage of the maximum rate observed in each experiment. The x axis shows the positionally defined amino acids (P1) as represented by the three-letter code.

Figure 2

 2 Figure 2 Overall fold of SplA.

Figure 3 .

 3 Figure 3. Atypical conformation of catalytic triad histidine.

Figure 4

 4 Figure 4 Putative binding mode of a consensus substrate to SplA.

Figure 5

 5 Figure 5 The role of N-terminal residue in the structures of mature SplA, V8 and chymotrypsin.

Table I . Experimentally determined SplA cleavage sites.

 I Residues corresponding to the consensus sequence are highlighted bold. Cleavage products of β-casein and carboxymethylated chicken lysozyme were identified by MS and Edman

	degradation, respectively.
	P4 Bovine β-casein P3	P2	P1	P1'
	I

Table II . Data collection and refinement statistics.

 II

	Biochemical Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081351
	1. Data collection		PDB ID 2w7u	PDB ID 2w7s
	Space group		P2 1 2 1 2 1	P2 1 2 1 2 1
	Cell constants:	a (Å)	73.99	72.62
		b (Å)	82.22	81.43
		c (Å)	134.19	132.02
	S) T/S S * Residues in parentheses constitute the template Consensus sequence W/Y L Y Fusion protein * (V) Y L Y (invariant region) of the CLiPS library and tested fusion proteins. Wavelenght (Å) B-Factor (Wilson) (Å 2 ) Resolution range (Å) 20-2.43 (2.56-2.43) 1.5418 29.2 Completeness (%) R merge (%) R meas (%) Observed reflections Unique reflections I/σ(I) Average multiplicity 98.2 (97.2) 7.5 (36.5) 8.5 (41.8) 146,329 (17,924) 30,882 (4403) 16.8 (4.1) 4.7 (4.1) 2. Refinement Resolution (Å) 20 -2.43 No. of reflection used 29,282 R-factor (%) 20.9 R free (%) 24.8 Average B (Å 2 ) 30.7 RMS from ideal values: Bond lenghts (Å) Bond angles (°) 0.006 0.632 Ramachandran statistics (%) Most favored regions Additionaly allowed regions Generously allowed regions 85.2 14.1 0.7 3. Content of asymmetric unit Average RMSD (C α ) of monomers (Å) 0.54 No. of protein molecules 4 No. of protein residues / atoms 797 / 6158 A c c e p t e d M a n u s c r i p t 1.0000 23.1 20-1.80 (1.90-1.80) 99.6 (100.0) 5.5 (21.6) 6.1 (24.0) 387,591 (56,106) 72,964 (10,566) 19.6 (6.5) 5.3 (5.3) 20-1.80 69,255 21.3 24.4 24.6 0.007 1.015 88.7 10.7 0.6 0.47 4 777 / 5861 No. of solvent molecules 36 247	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081351

© 2009 The Authors Journal compilation © 2009 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Biochemical Society

Biochemical Journal Immediate Publication. Published on 28 Jan 2009 as manuscript BJ20081351 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081351 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Biochemical Society

Acknowledgements:

We acknowledge Sui-Lam Wong from the University of Calgary, Canada, and Jozef Oleksyszyn and Ewa Pietrusewicz from Wrocław University of Technology, Poland, respectively for a kind donation of Bacillus subtilis expression strain and plasmid, and phosphonate inhibitors. This work was supported in part by funds of the Polish Ministry of Science and Higher Education (to JP), grants 13033 and N N301 032834 (to GD) and the European Social Fund and polish national budget in the framework of The Integrated Regional Operational Program (to GD). GD is also a recipient of The Foundation for Polish Science Scholarship for Young Scientists.


Enrico Girardi

Mary D Holdom

Anna M Davies

Brian J Sutton
email: brian.sutton@kcl.ac.uk

Andrew J Beavil

The crystal structure of rabbit IgG-Fc

Keywords: IgG, Fc fragment, immunoglobulins, glycosylation, rabbit IgG, immunoglobulin G, Fc, fragment crystallisable, Fab, antigen binding fragment, rmsd, root mean square deviation, MBP, mannose binding protein, RA, rheumatoid arthritis, RF, rheumatoid factor

We report the structure of the Fc fragment of rabbit IgG at 1.95 angstrom resolution. Rabbit IgG was the molecule for which Porter established the four-chain, Y-shaped structure of the antibody molecule, and crystals of the Fc ("Fragment crystallisable") were first reported almost 50 years ago in this journal (Porter R.R., 1959, Biochem. J., 73, 119-126). This high-resolution analysis, apparently of the same crystal form, reveals several features of IgG-Fc structure that have not previously been described. More of the lower hinge region is visible in this structure than in others, demonstrating not only the acute bend in the IgG molecule that this region can mediate, as seen in receptor complexes, but also that this region has a tendency to adopt a bent structure even in the absence of receptor. As observed in other IgG-Fc structures, the Cγ2 domains display greater mobility/disorder within the crystals than the Cγ3 domains; unexpectedly the structure reveals partial cleavage of both Cγ2 intra-domain disulphide bonds, while an alternative conformation for one of the cysteine residues in the intact bridge within the more ordered Cγ3 domains is observed. The N-linked oligosaccharide chains at Asn-297 are well-defined and reveal two alternative conformations for the galactose units on each of α(1-6) linked branches. The presence of this galactose unit is important for stabilising the structure of the entire branched carbohydrate chain, and its absence correlates with severity of autoimmune conditions such as rheumatoid arthritis in both human clinical studies and in a rabbit model of the disease. Rabbit IgG, through this highresolution structure of its Fc region, thus continues to offer new insights into antibody structure.

Introduction

The rabbit (Oryctolagus cuniculus) immunoglobulin gamma (IgG) was one of the first molecules of the immune system to be investigated, and the pioneering studies of Porter produced the very first information about the structure of the antibody molecule. Proteolytic cleavage of rabbit IgG by papain into three fragments, one of which crystallised [START_REF] Porter | The hydrolysis of rabbit gamma-globulin and antibodies with crystalline papain[END_REF] and was termed Fc (Fragment crystallisable), together with two identical fragments, termed Fab (Fragments antigen-binding), led to the first proposal of the four-chain, Y-shaped structure for IgG [START_REF] Fleischman | The arrangement of the peptide chains in gamma-globulin[END_REF].

Even before the hetero-tetrameric model of two identical heavy and two identical light chains was developed, the first immunoglobulin allotypes were described for the rabbit IGHG gene [START_REF] Oudin | The allotype of certain blood protein antigens[END_REF]. This observation led Oudin to hypothesize that IgG production was regulated by two independently segregating loci (named a and b). Several years later these two loci were identified, respectively, in the variable region of the heavy chain and the constant region of the kappa light chain genes, confirming the presence of two distinct protein chains in the molecule. Moreover, amino acid sequence studies on rabbit γ chains were pivotal in clarifying the nature of immunoglobulin domains [START_REF] Hill | Studies on the amino acid sequence of heavy chains from rabbit immunoglobulin G[END_REF] and the arrangement of disulphide bonds present in the molecule [START_REF] O'donnell | The disulphide bonds of the heavy chain of rabbit immunoglobulin G[END_REF]. Further enzymatic digestion and functional characterisation studies of rabbit IgG-Fc provided the first attempts to map the binding sites for complement and cytophilic activity [START_REF] Utsumi | Stepwise cleavage of rabbit immunoglobulin G by papain and isolation of four types of biologically active Fc fragments[END_REF][START_REF] Colomb | Characterization of a plasmin-digest fragment of rabbit immunoglobulin gamma that binds antigen and complement[END_REF][START_REF] Ovary | Biologic activities of rabbit immunoglobulin G in relation to domains of the Fc region[END_REF]. Later, the same molecule was used as a model for the determination of the composition and topology of the carbohydrate chains present on the Fc fragment [START_REF] Sutton | The three-dimensional structure of the carbohydrate within the Fc fragment of immunoglobulin G[END_REF][START_REF] Rademacher | Structural and conformational analysis of immunoglobulin-derived Nlinked oligosaccharides[END_REF], which play a role in stabilising the structure of the Fc and are required for binding to cell surface receptors.

Despite the key role played by this molecule in the history of immunology, no crystal structure is currently available for the Fc fragment of rabbit IgG. We describe here the 1.95Å resolution structure, one of the highest resolution Fc structures yet reported, from a crystal form that appears identical to the crystals pictured almost 50 years ago by Porter [START_REF] Porter | The hydrolysis of rabbit gamma-globulin and antibodies with crystalline papain[END_REF].

Experimental

Protein purification

Purified polyclonal rabbit IgG (Sigma-Aldrich, UK) was digested with papain according to the manufacturer's instructions (ImmunoPure Fab preparation kit, Pierce Corporation, USA). The Fab portion was removed from the resulting mixture using a protein A column. Fractions containing the Fc fragment and undigested IgG were then loaded onto a Superdex-200 gel filtration column (Amersham Pharmacia Biotech, UK) equilibrated with 0.5 M Tris pH 7.2, 0.25 M NaCl and 0.1% (w/v) sodium azide. The purity of the eluted sample was assessed by non-reducing SDS-PAGE; a single band indicated the presence of intact dimer (data not shown). Fractions containing the purified Fc were pooled and concentrated to 2 mg/ml at 4°C by centrifugation (Amicon, MWCO 10000, Millipore Corporation, USA) and stored at the same temperature until further use. acetate at pH 4.7 and 2 M sodium formate (reservoir) and 1 µl protein solution with an equal volume of reservoir (drop). The drops were stored at 18°C and plate-like crystals measuring approximately 0.1 mm by 0.1 mm typically appeared after two days. Their morphology was identical to those pictured [START_REF] Porter | The hydrolysis of rabbit gamma-globulin and antibodies with crystalline papain[END_REF][START_REF] Utsumi | Stepwise cleavage of rabbit immunoglobulin G by papain and isolation of four types of biologically active Fc fragments[END_REF] and characterised [START_REF] Poljak | Papain fragmentation of rabbit gamma-g-globulin: A crystallographic study[END_REF][START_REF] Aschaffenburg | Crystallographic studies of immunoglobulins: Crystallization of the Fc fragment of rabbit IgG with and without cleavage of the inter-chain disulphide bridge[END_REF] earlier.

Data collection and processing

Crystals were fished using nylon loops, soaked in a cryoprotectant solution (0.1 M sodium acetate, pH 4.7, 2 M sodium formate and 30% glycerol) and flash-cooled in liquid nitrogen. Data were collected at beamline ID29 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The data were processed with MOSFLM [START_REF] Leslie | Recent changes to the MOSFLM package for processing film and image plate data[END_REF], SCALA [START_REF] Evans | The CCP4 suite: Programs for protein crystallography[END_REF] and the CCP4 program suite [15]. The space group and cell parameters were found to be very similar to those observed previously [START_REF] Aschaffenburg | Crystallographic studies of immunoglobulins: Crystallization of the Fc fragment of rabbit IgG with and without cleavage of the inter-chain disulphide bridge[END_REF]. Calculation of the Matthews coefficient (2.53; 51.1% solvent) indicated the presence of one Fc molecule per asymmetric unit. Data collection and statistics are presented in Table 1. The structure was solved by molecular replacement with MOLREP [15,16] using protein atoms from the human IgG-Fc structure as a search model (PDB code 1H3T [17]). Refinement was performed initially with REFMAC [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] and later with PHENIX [START_REF] Adams | PHENIX: Building new software for automated crystallographic structure determination[END_REF]. TLS refinement was implemented using 10 TLS groups per chain, as suggested by the TLSMD server [START_REF] Painter | Optimal description of a protein structure in terms of multiple groups undergoing TLS motion[END_REF]. Cycles of refinement were alternated with rounds of manual model building with COOT [START_REF] Emsley | Coot: Model-building tools for molecular graphics[END_REF]. Protein atoms were modelled first, followed by carbohydrate, ligands and water molecules. The refined structure was analysed with COOT, PROCHECK [START_REF] Laskowsky | PROCHECK: A program to check the stereochemical quality of protein structures[END_REF] and MolProbity [START_REF] Davis | MolProbity: All-atom contacts and structure validation for proteins and nucleic acids[END_REF]. Secondary structure was assigned with the KSDSSP algorithm implemented in Chimera [24]. Omit maps were prepared in CNS [START_REF] Brunger | Crystallography & NMR system: A new software suite for macromolecular structure determination[END_REF]. Figures were prepared with PyMOL (www.pymol.sourceforge.net). Refinement statistics are presented in Table 2.

Atomic coordinates and structure factors have been deposited in the PDB database with the accession code 2VUO.

Results and discussion

Overall structure

The 1.95Å resolution structure of rabbit IgG-Fc displays the typical horseshoe-like arrangement of the Cγ2 and Cγ3 domains in the two polypeptide chains (Figure 1). Superimposition with the highest resolution human IgG-Fc structure (PDB code 1L6X; 73% sequence identity with rabbit IgG-Fc), revealed RMSD values of 0.81 and 0.76 Å for the two chains (performed with LSQMAN software [START_REF] Kleywegt | Use of non-crystallographic symmetry in protein structure refinement[END_REF]). Superimpositions of the individual domains (RMSD 0.37 and 0.47 Å for Cγ3; 0.80 and 0.85 Å for Cγ2) indicated that most of the differences between the rabbit and human structure occur in the Cγ2 domains.

In the rabbit IgG-Fc structure, interpretable density was present for residues Pro-230 to Ser-444 in chain A, and residues Pro-231 to Ser-444 in chain B; the three C-terminal residues were disordered. At the N-termini, although no density was observed in either chain for Cys-229, the residue involved in the inter-chain disulphide bond, much more of the "lower hinge" region is visible here than in other Fc structures (usually defined from Pro-238), except those complexed with receptors (FcγR) that bind to this part of the Fc molecule. However, Pro-230 and Pro-231 (in chains A and B respectively) are only 7.8Å apart (distance between closest atoms), consistent with an intact interchain disulphide bridge between residues Cys-229. In Figure 1, this lower hinge region of the polypeptide chain points towards the viewer, and demonstrates the flexibility and acute bend in the IgG molecule that it can mediate. It is striking that the conformation of this region in rabbit Fc is very similar to that observed in Fc-receptor complexes (PDB codes 1E4K, 1T89, 1T83) [START_REF] Sondermann | The 3.2-Å crystal structure of the human IgG1 Fc fragment-Fc gamma RIII complex[END_REF][START_REF] Radaev | The structure of a human type III Fc gamma receptor in complex with Fc[END_REF] and [START_REF] Saphire | Crystal structure of a neutralizing human IGG against HIV-1: A template for vaccine design[END_REF][START_REF] Harris | The three-dimensional structure of an intact monoclonal antibody for canine lymphoma[END_REF]. In the receptor complexes, a bend in this lower hinge region is demanded by the presence of the receptor. In the rabbit Fc structure, a single hydrogen bond between Glu-233 (chain B) and Arg-443 in the Cγ3 domain of a symmetry-related molecule may assist in stabilising this part of the structure, but the only other crystal packing contacts with any lower hinge region residues on either chain are a total of five van der Waals interactions (Glu-233 on both chains and Leu-235 on chain A; cut-off < 3.5Å). Thus although the presence of a symmetry-related molecule in this crystal form may restrict the conformational freedom of the lower hinge compared with other Fc crystal structures, it appears that this part of the hinge has a natural tendency to adopt this "bent" conformation even in the absence of bound receptor.

The Cγ2 domains are more mobile (or disordered) than the Cγ3 dimer, as reflected by their higher overall B factors (average values 27.8 and 21.2 Å 2 respectively, Table 2). This feature has been seen in almost all other Fc structures, from the first reported human IgG-Fc [START_REF] Deisenhofer | Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9-and 2.8-Å resolution[END_REF] to the recent highresolution structure 1L6X (average values 23.8 and 18.7 Å 2 respectively). No significant difference in the B values was observed between the two chains of rabbit IgG-Fc however, implying that the values for Cγ2 and Cγ3 reflect an intrinsic difference between the two domains, and are not the result of different crystal packing environments.

The conformation of the main chain for residues 283-296, adjacent to residue Asn-297 to which carbohydrate is covalently attached, has been found to adopt one of two different conformations in other IgG-Fc structures; these have been termed the 'classical' and the 'complex' conformation [START_REF] Matsumiya | Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1[END_REF]. In rabbit IgG-Fc, the structure conforms to the 'complex' conformation observed in the recently published high-resolution human IgG-Fc structures [17,[START_REF] Matsumiya | Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1[END_REF].

Intra-domain disulphide bonds

Each domain contains a conserved intra-chain disulphide bond, bridging the two β-sheets of the immunoglobulin fold. In both Cγ2 domains, the electron density indicates the presence of both an intact covalent bond and a broken state for the Cys-261 to Cys-321 bridge (Figure 2A). The differences in conformation between the two cysteinyl residues derives principally from a rotation around the χ 1 (N-Cα-Cβ-Cγ) torsion angle, and are almost certainly the result of radiation damage during data collection. In the Cγ3 domains there is no evidence of intra-domain disulphide bond cleavage, but Cys-425 in chain A (Figure 2B) displays two alternative conformations, indicating that some degree of flexibility is present even in the core of this immunoglobulin domain. The partial cleavage of both bonds within the Cγ2 domains is clearly consistent with the higher B values observed for this domain, as discussed above.

Allelic variation

Allotypes of the IGHG gene, which codes for the only IgG heavy chain subclass in the rabbit, have been described in the constant region of the γ heavy chain. They correlate with single amino-acid substitutions at positions 228 and 309 in the hinge and Cγ2 domain respectively [START_REF] Esteves | Hotspot variation at the CH2-CH3 interface of leporid IgG antibodies (Oryctolagus, Sylvilagus and Lepus)[END_REF]. Each locus presents two serologically defined alleles: d11 and d12 at position 228 and e14 and e15 at position 309. The two allelic variants at position 309, which is defined in the crystal structure, correspond to an Ala/Thr substitution. Given the nature of the rabbit IgG sample, no information was available regarding the sequence or the allelic proportions present in the crystallised protein. As the allelic variant with alanine is the more widespread [START_REF] Esteves | Restriction fragment alleles of the rabbit IGHG genes with reference to the rabbit IGHGCH2 or e locus polymorphism[END_REF], this residue (corresponding to the e15 allotype) was refined at position 309. No positive density in the F o -F c map (even at a contour level of ~2.0σ) was observed, suggesting that the allele with Ala was the one exclusively present in the crystal. The A c c e p t e d M a n u s c r i p t presence of an Ala residue at position 309 was later confirmed by mass spectrometry analysis (data not shown). An allotypic variant (nG4m; Leu/Val) is also found at position 309 in human IgG4 [START_REF] Brusco | Molecular characterization of immunoglobulin G4 gene isoallotypes[END_REF]. This amino acid is located at the Cγ2/Cγ3 interface, in the binding site for the neonatal Fc receptor [START_REF] West | Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor[END_REF]. While the two variants characterised in human Fc both code for hydrophobic residues, the e14 allotype in rabbit Fc codes for a polar residue, which may affect the binding of the neonatal receptor, although there are no reported functional studies that test this hypothesis. Incidentally, we observed positive density on both chains at Ala-396 indicative of a threonine or valine residue, although no variability has been noted previously at this position.

Glycosylation

Rabbit IgG-Fc possesses a conserved glycosylation site at Asn-297 in each Cγ2 domain, at which a complex biantennary type oligosaccharide is covalently attached. The carbohydrate covers a hydrophobic patch on the Cγ2 domain surface and extends into the cavity between the two Cγ2 domains (Figure 3). Its presence stabilises and maintains the structure of the protein, as proposed initially from studies of the human IgG-Fc fragment [START_REF] Deisenhofer | Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9-and 2.8-Å resolution[END_REF] and later confirmed by microcalorimetry experiments [START_REF] Ghirlando | Glycosylation of human IgG-Fc: Influences on structure revealed by differential scanning micro-calorimetry[END_REF]38] and structural studies of sequentially deglycosylated Fc glycoforms [17]. Moreover, the conserved oligosaccharide plays a crucial role in IgG effector functions: it is required for optimal Fcγ receptor binding, and mediates interactions with serum lectins such as mannose binding protein (MBP) that leads to complement activation [39,[START_REF] Malhotra | Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein[END_REF].

While the core of the oligosaccharide is conserved, heterogeneity is observed in the fucosylation (found in ~30-40% of rabbit IgG-Fc molecules), galactosylation (~50-60%) and sialylation of the chain (~25%); an additional bisecting N-acetylglucosamine may also be present (~25-30% of rabbit IgG-Fc molecules) [START_REF] Hamako | Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species[END_REF][START_REF] Raju | Species-specific variation in glycosylation of IgG: Evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics[END_REF]. On both chains, carbohydrate units up to and including the galactose at the end of each of the α(1-6) branches (Gal-6) were built into good electron density (Figure 3), whereas on the α(1-3) branches, only Man-7 and GlcNAc-8 (the latter only on chain C, Figure 3) were built. The lack of interpretable electron density for fucose, terminal sialic acid or a bisecting N-acetylglucosamine is consistent with the low fractional compositions reported. The α(1-3) branches extend into the area between the two Cγ2 domains and lie close to each other, but none of the contacts between the two chains described previously in the much lower resolution analysis [START_REF] Sutton | The three-dimensional structure of the carbohydrate within the Fc fragment of immunoglobulin G[END_REF] were seen. While some unliganded human Fc structures show inter-chain carbohydrate contacts, others do not [START_REF] Oganesyan | Structural characterization of a mutated, ADCC-enhanced human Fc fragment[END_REF]; whether or not such contacts occur appears to depend upon domain quaternary structure, which in turn may be determined by crystal packing contacts. The α(1-6) branches lie close to the first two strands (A and B) of the C γ 2 domain, their core regions interacting with residues Phe-241, Phe-243 and Glu-265.

The extent of galactosylation in IgG molecules is highly variable, with a reported 40-50% of the molecules entirely lacking galactose in healthy rabbits [START_REF] Hamako | Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species[END_REF][START_REF] Raju | Species-specific variation in glycosylation of IgG: Evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics[END_REF]. We observed density at the end of each α(1-6) branch, clearly indicating the presence of a galactose residue (Gal-6). When refined with an occupancy of 100% (as for all the other carbohydrate units), higher B factors and some negative difference electron density indicated a slightly lower occupancy and/or the presence of alternative conformations. (Refinement with occupancy values set between 40 and 60% revealed positive difference electron density). It was clear however, that the principal conformation adopted by Gal-6 is different in the two chains (Figures 1 and3). In chain A, the O6 atom of Gal-6 points away from the Cγ2 domain, while in chain B, the O6 atom points towards the Cγ2 surface. These two conformations are related by an approximately 180° rotation of the plane of the hexose ring, but in both, Gal-6 lies in the same cavity, forming (different) hydrogen bonds to the same residues Pro-244, Lys-246 (although density for CE and CZ of Lys-246 on chain A is ill-defined), Glu-258 and Thr-260. The galactose conformation in chain A is identical to that seen in the 1.65Å human IgG-Fc (PDB code 1L6X), but no asymmetry was observed in that structure as the two chains are related by There is thus some variability in the nature of the interaction between this carbohydrate unit and the protein surface.

Fc galactosylation and functional implications

The galactose content of human IgG-Fc has been shown to correlate inversely with disease progression in rheumatoid arthritis (RA) [START_REF] Parekh | Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG[END_REF][START_REF] Axford | Glycosylation and rheumatic disease[END_REF] and other auto-immune diseases [START_REF] Cremata | Hypogalactosylation of serum IgG in patients with coeliac disease[END_REF]. Various mechanisms have been proposed: the absence of galactose may expose hydrophobic surfaces that promote Fc-mediated IgG aggregation, or generate new Fc epitopes that are recognised by rheumatoid factor (RF) autoantibodies [START_REF] Newkirk | Rheumatoid factor avidity in patients with rheumatoid arthritis: Identification of pathogenic RFs which correlate with disease parameters and with the gal(0) glycoform of IgG[END_REF]. The galactose unit is also known to be a key determinant for stabilising the carbohydrate chain's interaction with the protein domain. Its absence leads to greater mobility of the chains as shown by NMR [START_REF] Wormald | Variations in oligosaccharide-protein interactions in immunoglobulin G determine the sitespecific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides[END_REF] and crystallographic [START_REF] Corper | Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction[END_REF] studies, and this enhanced accessibility permits interaction with lectins such as MBP, leading to complement activation [START_REF] Malhotra | Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein[END_REF]. Similar changes in the galactose content of rabbit IgG have been reported after hyperimmunisation, and enhanced avidity of rabbit IgG RF autoantibodies for IgG-Fc was shown to be due to decreased galactose content [START_REF] Murray | Evidence that the Fc region of autologous rabbit IgG isolated before and after hyperimmunization is structurally different: Recognition by rheumatoid factor and monoclonal antibodies[END_REF]. In a rabbit model of RA, a long-term immunisation study that followed RF titre, avidity and IgG-Fc galactose content during disease progression showed the same inverse correlation between RF avidity for IgG-Fc and galactose content [START_REF] Ciric | A longitudinal study of the relationship between galactosylation degree of IgG and rheumatoid factor titer and avidity during long-term immunization of rabbits with BSA[END_REF]. The crystal structure of rabbit IgG-Fc has revealed a conformational variability in the mode of interaction of this key carbohydrate residue and the Cγ2 domain, underlining the tenuous nature of this contact and its role in tipping the balance between a mobile carbohydrate chain and an ordered structure in contact with the Cγ2 domain surface.

Rabbit IgG provided the very first insights into antibody structure, and through the high-resolution crystal structure of its Fc region reported here, it continues to reveal new structural details and enhance our understanding of the antibody molecule.

Tables

Figure 2 Conformations of the intra-domain disulphide bridges

Stereo images of the region surrounding the intra-chain disulphide bridges in Cγ2 (Figure 2A) and Cγ3 (Figure 2B) of chain A. The electron density is calculated from a composite simulated annealing omit map, contoured at 1σ, generated omitting 4% of the structure per cycle. Carbon atoms are represented in green, oxygen in red, nitrogen in blue and sulphur atoms in orange. The highly conserved tryptophan residues (277 and 381) adjacent to the disulphide bonds can be seen towards the back of the image. In Cγ2 (Figure 2A) the disulphide bond is partially broken, while in Cγ3 (Figure 2B), alternative conformations for the intact bridge are observed.

Biochemical

  Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20081355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081355 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society in intact immunoglobulins (1HZH, 1IGT)

Biochemical

  Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20081355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081355

Biochemical

  Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20081355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081355 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society a crystallographic dyad.

Figure legends Figure 1

 legends1 Figure legends

Figure 3

 3 Figure 3The carbohydrate structure of rabbit IgG-Fc A) Electron density for the N-linked oligosaccharides at Asn-297 in each polypeptide chain, calculated from a composite simulated annealing omit map (contour level: 1σ), is shown together with a representation of the two Cγ2 domains. B) Schematic representation of the complex biantennary oligosaccharide chain present in rabbit IgG-Fc. The residues modelled in the electron density are highlighted in light grey (GlcNAc-8 is only modelled on chain C).

Table 1 : Data collection and processing statistics

 1

	Beamline

Table 2 :

 2 Refinement statistics

	Resolution range (Å)	56.7-1.95
	No. of: protein atoms	3479
	carbohydrate atoms	186
	water molecules	398
	formate ions	6
	azide ions	2
	glycerol molecules	4
	Average B factors (Å 2 )
	protein	24.6
	carbohydrate	42.5
	water molecules	35.1
	formate ions	39.9
	azide ions	38.3
	glycerol molecules	48.5
	protein Cγ2 (chain A)	28.5
	protein Cγ2 (chain B)	27.0
	protein Cγ3 (chain A)	21.8
	protein Cγ3 (chain B)	20.5
	R cryst (%)	16.9
	R free (%) (5% of reflections)	20.4
	rmsd bond lengths (Å)	0.004
	rmsd bond angles (°)	0.928
	Ramachandran plot (% of total	favoured: 99.5, allowed: 0.5,
	residues)	disallowed: 0

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We are grateful to Sophia Karagiannis for assistance with the Fc fragment preparation and to Roberto Steiner, Stella Fabiane, Balvinder Dhaliwal and Silviana Comsa for helpful discussions. We are also grateful to Karen Homer (Dental Institute, King's College London) for the mass spectrometry analysis. We thank the staff at ID29 at ESRF (Grenoble, France) for assistance with data collection. E.G. is funded by a King's College London Strategic Development Studentship. Biochemical Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20081355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081355 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society