Giulia Maria Stella

Silvia Benvenuti

Daniela Gramaglia

Aldo Scarpa

Anna Tomezzoli

Paola Cassoni

Rebecca Senetta

Tiziana Venesio

Ernesto Pozzi

Alberto Bardelli

MD Paolo M Comoglio
email: pcomoglio@gmail.com

John Wiley

MET mutations in cancers of unknown primary origin (CUPs)

Keywords: Met, tyrosine kinase, somatic mutation, invasive growth, metastases

Introduction

It is widely accepted that systemic neoplastic spread is a late event, resulting from accumulation of genetic alterations occurred in local progression [START_REF] Fearon | A genetic model for colorectal tumorigenesis[END_REF]. However, in some instances, distant dissemination arises at a very early stage, so that metastases reach clinical relevance before primary lesion (Hüsemann et al., 2008;Weinberg, 2008). In a number of cases, integration of immunohistochemistry [START_REF] Engelman | MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling[END_REF] and sophisticated imaging and radio-metabolic techniques (Regelink et al., 2002;[START_REF] Sève | The role of 2-deoxy-2-[F-18]fluoro-D glucose positron emission tomography in disseminated carcinoma of unknown primary site[END_REF] helps in identifying the primary tumor site (Pentheroudakis et al., 2007); nevertheless sometimes metastases remain really 'orphan'. The latter defines a highly malignant syndrome known as cancer of unknown primary origin (CUP) which may display undifferentiated phenotype. These tragic cases may be described as not only of unknown origin, with respect to organ site, but also of unknown pathogenesis. Representing a still unsolved clinical problem lacking effective therapeutic regimens, CUPs describe a specific entity that occurs with an incidence that cannot be ignored, 3-5% of all human cancers [START_REF] Briasoulis | Cancers of unknown primary site: ESMO clinical recommendations for diagnosis, treatment and follow up[END_REF].

On the other hand, an increasing body of evidence suggests that the invasive malignant phenotype requires the aberrant execution -in time and space-of a physiological genetic program named 'invasive growth', resulting from the integration of different biological activities, including cell-cell dissociation ('scatter'), migration, invasion and cellular proliferation. The program is driven by specific factors, among which Scatter Factor, also known as Hepatocyte Growth Factor (HGF), and semaphorins [START_REF] Trusolino | Scatter-factors and semaphoring receptors: cell signaling for invasive growth[END_REF].

The receptor for Scatter Factor/HGF is encoded by the MET oncogene (NM_000245.2) [START_REF] Boccaccio | Invasive growth: a MET-driven genetic programme for cancer and stem cells[END_REF], located on chromosome 7q31, made of 21 exons codifying for a trans-membrane tyrosine kinase protein. The extracellular region contains a 500 aminoacid SEMA domain, distinctive of the semaphorin superfamily [START_REF] Comoglio | Scatter factors and invasive growth[END_REF][START_REF] Tamagnone | Plexins are a large family of receptors for transmembrane, secreted and GPI-anchored semaphorins in vertebrate[END_REF], involved in receptor dimerization and activation [START_REF] Gherardi | Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor[END_REF]Kong-Beltram et al., 2004); a cysteine-rich domain (known as MET-Related Sequence -MRS); and a protein-protein interaction site made of four immunoglobulin-like structures (IPT domain). The intracellular portion of the receptor is made of a juxtamembrane region; a catalytic site containing two tyrosines (Y1234 and Y1235) which controls the enzymatic activity and a C-terminal regulatory tail with two tyrosines (Y1349 and Y1356) that, upon phosphorylation, create a docking site for intracellular signal transducers. The latter is responsible for the concomitant activation of multiple intracellular signaling pathways, triggering a cascade of biological responses, ultimately leading to the invasive phenotype (Ponzetto et al., 1994). In human cancers MET activation confers a selective advantage for tumor progression [START_REF] Engelman | MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling[END_REF]). It generally occurs as a late event, mainly consequent to receptor overexpression driven by unfavourable micro environmental conditions -e.g. hypoxia (Pennacchietti et al., 2003)-; in some instances overexpression is due to gene amplification [START_REF] Comoglio | Drug development of MET inhibitors: targeting oncogene addiction and expedience[END_REF]. Somatic point mutations are rarely found, accounting for no more than 3-4% of unselected primary cancers (COSMIC database: http://www.sanger.ac.uk). We hypothesized that MET activation by somatic mutations at early stages of cancer onset might sustain aberrant execution of the invasive growth program and result in precocious metastatic phenotype, such as that identifying CUPs.

Materials and Methods

CUP patients selection.

We analyzed 47 specimens from a cohort of cancer patients in which the first manifestation detected were two or more metastases. Formalin-fixed paraffin-embedded (FFPE) samples derived from the two different institutions: Department of Pathology at University of Verona and Department of Pathology at University of Torino. The majority of collected samples were obtained from biopsies; brain metastases derived from surgical specimens. Clinical data are listed in Supp. Table 1. All patients received a thorough sequential multistep diagnostic work-up following the European Society of Medical Oncology (ESMO) guidelines [START_REF] Briasoulis | Cancers of unknown primary site: ESMO clinical recommendations for diagnosis, treatment and follow up[END_REF]. Diagnosis of CUPs was reached in those patients with no detectable site of primary origin, after exhaustive anamnesis and diagnostic evaluation including complete physical examination, complete laboratory tests, whole body CT scan and FDG-PET. The immunohistochemical profile was defined by a panel of at least ten markers, according to accepted procedures outlined by Horlings et al. (Horlings et al., 2008) and further refined by Rossi et al. (Rossi et al., 2009).

Immunohistochemistry (IHC).

As mentioned above, given that the accuracy of IHC is a crucial step in CUP diagnosis, immunophenotype of each case was carefully validated according to accepted standards as reported by Horlings et al. (Horlings et al., 2008) and by Rossi et al. (Rossi et al., 2009). Panels of up to 10 antibodies were used on FFPE tumor sections, according to the manufacturer's recommendations. The gastric origin of the metastatic lesions was recognized by the co-expression of various immunohistochemical markers, such as cytokeratins 7 and 20 and CDX2.

Metastatic lesions from the biliary-pancreatic region proved to be positive for cytokeratin 19. Immunohistochemical reactions were evaluated independently by two pathologists; controversial cases were reevaluated jointly until a consensus was reached.

Genetic analysis.

PCR primers designed to amplify the entire coding sequence of MET (NM_000245.2) and RON (NM_002447.2), KRAS (NM_004985.3) exon 2, EGFR (NM_005228.3) exons 18-19-20-21 and PIK3CA (NM_006218.2) exons 9-20 are listed in Supp. Table S2. A total of 1216 PCR products were generated from independent aliquots of DNA and subjected to direct sequencing. The somatic origin of each mutation was confirmed by sequencing, together with neoplastic DNA, normal DNA obtained by laser capture microdissection of not-transformed areas adjacent to the neoplastic lesions, whereas, in the case of small samples, normal DNA was extracted from different histological specimens of the same patient (archived at Pathological Banks), as previously described (Moroni et al., 2005). Mutations were detected only in transformed tissues. Microsatellite instability (MSI) was assessed by using the panel of repeats as previously described [START_REF] Suraweera | Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR[END_REF]. These five mononucleotide markers (BAT25, BAT26, NR21, NR24 and NR27) were amplified according to the modifications proposed by Burhard et al. (Burhard et al., 2006), and then analyzed on ALFexpress II Sequencer (GE Healthcare Europe). FISH analysis was carried out on 4 µm FFPE, as already described [START_REF] Casorzo | Fluorescence in situ hybridization (FISH) evaluation of chromosomes 6, 7, 9 and 10 throughout human melanocytic tumorigenesis[END_REF], using the following commercially available DNA probes: CEP 7 locus D7Z1-SpectrumGreen™ and locus-specific LSID7S522-SpectrumOrange™probe for band 7q31.1 and LSI-EGFR-SpectrumOrange™probe for band 7p12 (Vysis/Abbott Molecular, IL, USA).

MET mutant expressing constructs.

MET mutations p.Cys385Tyr (g.1154G>A) and p.Val1312Ile (g.3932G>A) were generated using QuickChange Site-Directed Mutagenesis XL kit (Stratagene, LaJolla) according to the manufacturer's instructions and confirmed by direct sequencing. The entire sequence of all engineered cDNAs was checked and no other unexpected mutations were found. cDNAs, either wild-type or mutated, were then cloned into P156RRLsinPPThCMVMCSpre lentiviral vector. COS-7 and T47D (from ATCC) were cultured in either in DMEM or RPMI-1640 media (Invitrogen Carlsbad, CA), supplemented with 10% FCS (Sigma Aldrich, ST.Louis, MO). Viral particles were produced by transient co-transfection of 293T cells with 10 µg PRRL2-WT-MET; PRRL2-p.Cys385Tyr-MET, PRRL2-p.Val1312Ile-MET in combination with 3 µg of envelop plasmid (pMD2-VSV-G), 5 µg of core packaging plasmid (pMDLg/pRRE) and 2,5 µg of pRSV-REV as previously described [START_REF] Vigna | Lentiviral vectors: excellent tools for experimental gene transfer and promising candidate for gene therapy[END_REF]. Expression of wild type Met, p.Cys385Tyr-Met and p.Val1312Ile-Met was tested by Western Blot analysis. Antibodies used are listed below. Anti MET: DQ-13 and DL-21 (Prat et al., 1991); C-12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibodies against the MET phosphor-epitopes [PY1349] and [PY1234/1235] were from BioSource International; anti actin from Santa Cruz. Conjugated secondary antibodies were from Amersham (Arlington Heights, VA).

Biological assays.

Cell proliferation assays, either in absence or in presence of HGF (50 ng/mL), were performed using ATP-lite Luminescent Assay System (PerkinElmer Life And Analytical Sciences, Inc., MA, USA). The experiments were repeated three times each one performing triplicates. Anchorage-independent growth was assayed plating 5X10 3 cells in 0,5% agarose. HGF was supplied in the overly medium (40 ng/mL). Cell viability was assessed by Alamar blue staining (Resazurin sodium salt, Sigma-Aldrich, St.Louis, MO), then colonies were visualized using 0.02% iodonitrotetrazolium chloride (0.02% in PBS, Sigma-Aldrich, St.Louis, MO) and counted at a DMIL microscope (Leica) equipped with a digital camera (DFC320; Leica). For the wound healing assay, confluent cell layers were starved for 24 hours in 2% FCS. The assay was performed as previously described (Michieli et al., 2004) and quantified by time-lapse microscopy (one image every twenty minutes, for thirty-six hours), plotting the distance between the scratch margins calculated as mean of ten different measurements expressed as percentage of the initial distance. When required, medium was supplemented with HGF. For invasion assays, 5X10 4 cells were seeded in Transwell permeable supports (Cole-Parmer Vernon Hills, Illinois 60061) with the upper side of the porous polycarbonate membrane coated with 9,8 µg/mL Matrigel (BD Biosciences, Franklin Lakes, NJ USA). Assays were carried out in presence of 2% FCS, with or without 20 ng/mL HGF, for 24 hours. Migrated cells were stained with crystal violet, then detected using a microscope (DMIL; Leica) equipped with a digital camera (DFC320;Leica), and photographed with GIMP 2.2.8 software (The GIMP-GNU Image Manipulation Program) and quantified using Image J software.

Results

Patients selection.

Forty-seven cancer patients in which the first manifestation detected were two or more metastases were selected as described in the method section. Clinical data are listed in Supp. Table S1. The diagnosis of 'early metastatic cancer' was reached in those patients with no detectable site of primary origin after exhaustive anamnesis and diagnostic evaluation, including physical examination, complete laboratory tests, whole body CT scan and FDG-PET. Based on the immunohistochemical (IHC) profile it was possible to identify the putative site of origin in 24 patients, while the remaining 23 cases were classified as 'truly' cancers of unknown primary origin (CUPs). The putative primary suggested by IHC was more often lung (50%) followed by liver-biliary tract or pancreas (25%): these tissue-of-origin profiles are coherent with published data obtained at autopsy (Pentheroudakis et al., 2007;[START_REF] Varadhachary | Molecular profiling of carcinoma of unknown primary and correlation with clinical evaluation[END_REF]. The two cohorts displayed the typical early metastatic cancer clinical characteristics, even though CUPs featured a more aggressive behaviour. Predominant histopathology was adenocarcinoma in the cohort with solved primary (70,8%) and undifferentiated carcinoma in CUPs (78,2% ).

MET mutations in CUPs.

In the overall population of early metastatic cancers MET (MIM# 164860) was found mutated in 7 out of the 47 (~15%) samples analyzed. All mutations found in this study have been validated by processing independent aliquots (extracted from the same paraffin block at different times) of tumor DNA in multiple PCR amplification and sequencing rounds. The somatic origin of the mutations was confirmed by processing, together with tumor DNA, normal matched DNA obtained by laser capture micro dissection of not-transformed areas adjacent to the neoplastic lesions. Mutations were found only in tumor specimens, while normal matched DNA showed wild type MET. Mutations were confirmed by sequencing tumor genomic DNA both in forward and reverse directions. Surprisingly all mutations occurred in undifferentiated carcinomas with no identifiable tissue of origin (Supp. Table S1). In this 'truly' CUP cohort the overall occurrence of MET mutations is extremely high (30%). Since mutations occur as digital events (mutation absence/presence), the binomial distribution represents the statistical function that correctly describes this phenomenon [START_REF] Taylor | An Introduction to Error Analysis[END_REF]. By its simple application to data on MET mutations reported by COSMIC database (http://www.sanger.ac.uk) the results become relevant in respect to the unselected cancer population in which MET is mutated in 4% of cases.

The incidence is noteworthy even if compared to that of upper aero-digestive track tumors, which actually display the highest registered mutational rates (14%, data from COSMIC Database). In both cases the probability of the event (mutation) occurrence in a population of 47 samples, would be strictly near to zero; indeed the value obtained in fully CUPs (7 cases) hugely exceeds the expected one (Supp. Fig. S1).

Four somatic mutations (p.Gln142X; p.His150Tyr; p.Glu168Asp and p.Cys385Tyr) clustered in exon 2, which codifies for the SEMA domain, one mutation (p.Thr1010Ile) was found in exon 14, which codifies for the juxtamembrane domain and one (p.Val1312Ile) affected a residue located in exon 20, encoding for the tyrosine kinase domain, nearby the active site (Fig. 1). Two out of six mutations found in this study (p.Glu168Asp and p.Thr1010Ile) have already been described ( Di [START_REF] Renzo | Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas[END_REF]Ma et al., 2008;[START_REF] Seiwert | The MET receptor tyrosine kinase is a potential novel therapeutic target for head and neck squamous cell carcinoma[END_REF]; the other four are novel changes. As reported above, these results were validated through detection of the same mutation by processing independent aliquots of tumor DNA in multiple PCR amplification and sequencing rounds; moreover no mutations were found in matched DNA extracted from adjacent normal tissue.

In order to rule out the possibility of high mutational background due to repair deficiency or environmental exposure, we performed standard microsatellite instability (MSI) assays. We analyzed six out of seven MET mutated patients (lacking material from the seventh) and none of them showed MSI (Table 1). To further support this result we checked in the same cases the mutational profile of 'hot spot' regions of three oncogenes frequently mutated in cancers: KRAS (MIM# 190070) exon 2, EGFR (MIM# 131550) exons 18-19-20-21 and PIK3CA (MIM# 171834) exons 9-20 (Table 1); no mutations were found. As further control, we also checked the full-length coding sequence of RON (MIM# 600186). The RON gene, located on chromosome 3p21.3, has a similar size to MET, being made by 20 exons. Ron belongs to the Scatter Factor receptor family encoding a receptor which is similar to Met (63% homology; Ronsin, 1993), featuring an extracellular portion containing a SEMA domain, a transmembrane region and an intracellular tyrosine kinase domain. In four out of the seven MET mutated patients the cancer genomic DNA available was enough to sequence the whole RON gene; in other two cases the analysis was limited to the exons encoding for the SEMA and the tyrosine kinase domains.

In none of the CUP patients harbouring MET mutations, RON was found to be mutated. We also analyzed MET amplification was never detected (some samples carrying an increased MET copy number in consequence to a low grade of chromosome 7 polysomy, Table 1). To further investigate if other genes -frequently mutated in cancers-could be the driving force in CUP patients harbouring MET wild type, we performed the mutational analysis of the tree 'hot spot' oncogenes : KRAS exon 2; EGFR exons 18-19-20-21 and PIK3CA exons 9-20. No mutations were detected. On the contrary, four mutated samples were detected in the non-CUP group. Two cases (ID samples 13 and 19) carried EGFR somatic changes, p.Leu858Arg and c.2184_2283del respectively.

The other two mutated cases (ID samples 21 and 32) featured the KRAS Gly12Asp activating mutation. The above data strengthen the selective association between the CUP phenotype and MET, but not KRAS, EGFR or PIK3CA.

Within the limits of the small cohort analyzed in this study, the median survival of patients harbouring MET-mutations was of 8 months while that of MET-wild type patients 13 months (Supp. Fig S2). Taken together these data suggest a potential role of MET mutational profile in CUPs as negative prognostic marker.

Functional characterization of MET mutations.

We focused on the novel nucleotide changes identified, and among them the p.Cys385Tyr and the p.Val1312Ile mutations, localized in the SEMA or the tyrosine kinase domain, respectively. Their oncogenic potential was investigated by reproducing the mutations in cDNAs and expressing the mutated Met proteins in two different mammalian cell lines, T47D and COS-7. All experiments were performed in both cell lines, obtaining comparable results. Notably T47D do not express endogenous Met. Lentiviral expression of wild type receptor was used as a control. We investigated receptors phosphorylation and downstream signaling. We demonstrated that p.Val1312Ile (TK mutant) was constitutively phosphorylated, both on the catalytic domain (Y1234 and Y1235) and the docking site tail (Y1349, Fig. 2). Results were confirmed by in vitro kinase activity assay (data not shown). The two major downstream effectors of METt signaling, AKT and ERK, were coherently persistently activated (data not shown). On the other hand, as reasonably expected, the p.Cys385Tyr receptor (SEMA mutant) was not basally phosphorylated (Fig. 2). However, as described below, both mutations were able to activate the invasive growth response.

We analyzed the key features of the invasive growth phenotype: proliferation, motility, invasion and anchorage-independent growth (Fig. 3). The TK mutant (p.Val1312Ile) showed an increase in proliferation rate, as well as -unexpectedly-the SEMA mutant (p.Cys385Tyr). Cell proliferative rates were also tested upon HGF F o r P e e r R e v i e w 10 stimulation and were further increased in both mutants (Fig. 3, panel A). Cell motility was assessed by a cell monolayer wound healing assay, both in basal conditions and upon ligand stimulation. We observed that TK mutant featured an enhanced motile phenotype when compared to wild type Met. 'Healing speed' increased about two-hundred folds in basal conditions and two-hundred and fifty folds upon HGF stimulation in p.Val1312Ile. The SEMA mutant behaved, in wound healing assay, as the wild type receptor, whereas significantly augmented upon HGF stimulation (Fig. 3, panel B). As invasive behaviour of cancer cells is reflected, in vitro, by their ability to migrate through matrigel-coated Boyden chambers [START_REF] Terranova | Use of a reconstituted basement membrane to measure cell invasiveness and select for highly invasive tumor cells[END_REF] we performed a matrigel invasion assay and showed that the invasive capacity was significantly increased by transduction of cDNAs harbouring either mutations (p.Val1312Ile and p.Cys385Tyr), both in basal conditions and upon HGF stimulation. TK mutant displayed a hyper-invasive behaviour, which was further enhanced in the presence of a ligand gradient. SEMA mutant's phenotype appeared to be less aggressive but still significantly higher (p<0.005) compared to wild type Met (Fig. 3,panel C). The clonogenic properties of Met mutants were assessed by anchorage independent growth assay (Fig. 3, panel D). TK mutant showed a significant increase (p<0.001) in number of colonies formed. Interestingly clonogenic growth was enhanced at high degree in cells expressing the SEMA mutant. Anchorage-independent growth of both mutants was significantly augmented by HGF supplementation. Although these in vitro data do not provide incontrovertible proof supporting the key role of the described mutations in generating the hyper-metastatic phenotype, they at least provide evidence that the Met receptors mutated in CUPs were functional. Mutations occur preferentially in hereditary and sporadic papillary renal cancers (Schmidt et al., 1997), gastric and childhood hepatocellular carcinomas (Lee et al., 2000;Park et al., 1999), thoracic neoplasms (lung cancers and malignant pleural mesotheliomas, [START_REF] Cipriani | MET as a target for treatment of chest tumors[END_REF] as well as melanomas (Puri et al., 2007). Activation of MET by mutations occurs as a late event, and it has been associated with the acquisition of metastatic potential. For instance it has been demonstrated that, during progression toward metastasis, head and neck carcinomas select cells harbouring MET mutations (Lorenzato et al., 2002), On the other hand, in the vast majority of tumors, MET activation is due to over-expression : the event exacerbates malignancy by usurping the (otherwise physiological) anti-apoptotic and pro-invasive properties of the proto-oncogene, as an 'expedience' to gain fitness under adverse micro-environmental conditions (for a review see [START_REF] Comoglio | Drug development of MET inhibitors: targeting oncogene addiction and expedience[END_REF].

We now demonstrate that MET mutations occur with remarkable high frequency (30%) in metastatic cancers of unknown primary origin (CUPs). The relatively high incidence of activating MET mutations found in CUPs suggests the involvement of the oncogene in this still obscure pathology. Within the limits of the cohort analyzed in this study, MET mutational profile in CUPs clearly classifies MET mutated patients as those displaying the most aggressive disease. It should be noted, however, that the CUP group is enriched for tumors lacking differentiation markers, while the non-CUP group is enriched for specific tumor types, and thus the two groups are biased for differentiation grade and organ of origin. Therefore, MET mutation may as well reflect either differentiation grade and/or organ of origin. In this respect a preferential expression of MET in cancer stem cells has been postulated [START_REF] Boccaccio | Invasive growth: a MET-driven genetic programme for cancer and stem cells[END_REF]. However, this report provides experimental evidence that the mutations found in CUPs are functional, as their transduction in reporter cells 'in vitro' confers a clear invasive growth phenotype.

The mutations detected in CUPs, affect both the tyrosine kinase (catalytic) domain (p.Val1312Ile) as well as the extracellular SEMA domain (p.Gln142X; p.His150Tyr; p.Glu168Asp and p.Cys385Tyr).

Interestingly the three mutations affecting the SEMA domain of the receptor have been found in hematogenous metastases to the brain, a sanctuary site that can be reached only by most aggressive clones, capable to cross the blood-brain barrier. While it has been extensively demonstrated that mutations activating the kinase are tumorigenic (Michieli et al., 1999;Graavel et al., 2004), the oncogenic activity of the SEMA mutations was not fully predicted. This unexpected behaviour suggests that the non-catalytic domain of the MET receptor might be somehow involved in tumor progression. Although the mechanistic explanation of such a phenotype goes behind the scope of this report, we can speculate a yet unknown role in the control of the invasive growth program of the SEMA domain, a protein-protein interaction motif. Mutations localized within the SEMA domain might interfere either with the ligand binding or with the three-dimensional structure of the receptors.

Several therapeutic strategies have been developed to block MET signalling, each focusing on one of the sequential steps that regulates MET activation [START_REF] Comoglio | Drug development of MET inhibitors: targeting oncogene addiction and expedience[END_REF]. To date, dozens of small inhibitors and a few monoclonal antibodies have been actively pursued to antagonize MET activation. Based on experiences from EGFR inhibition, the occurrence of genetic alterations of MET activity might induce direct oncogene addiction and therefore predict therapeutic responsiveness. In addition to canonical mutations occurring within the kinase region, the described novel mutations affecting the SEMA domain may help identifying the responsive patient subset for personalized anti-MET therapy. Overall, the above described mutations have two clinical implications for the CUPs -still an orphan disease-providing: (i) a potential functional marker, with diagnostic and prognostic implications; (ii) a rationale for clinical trials with MET inhibitors. * denotes statistically significant differences.

F o r P e e r R e v i e w

6

  Lentiviral cell infection and Western blotting.

  gene copy number (by fluorescent in situ hybridization-FISH) in the MET mutated patients and MET

  point mutations is a (relatively) rare event, occurring in 3% of all cancer types.

•••Figure 1 .

 1 Figure LegendsFigure 1. MET mutations in CUPs. Met receptor structure displaying mutations found in CUPs. § PM: plasma membrane. *P: sites of phosphorylation. MRS: MET Related Sequence.• IPT: protein-protein interaction site.

Figure 2 .

 2 Figure 2. MET mutations phosphorylation status. Receptors phosphorylation was assessed in basal conditions and upon HGF stimulation in a time-course experiment (5, 10 and 20 minutes). The panel shows T47D extracts expressing wild type Met (MET WT), Met harboring the SEMA (p.Cys385Tyr) or the TK (p.Val1312Ile) mutations. Blots were decorated with antibodies against pospho-Met proteins (Y1234-1235 and Y1249), and total Met protein (DL21).

Figure 3 .

 3 Figure 3. The invasive growth phenotype of TK (p.Val1312Ile) and SEMA (p.Cys385Tyr) MET mutants.Results are means +/-SD. Comparisons were made using a two-tailed Student's t-test. P-values less than 0.05 were considered to be statistically significant.A) Cell proliferation of T47D expressing wild type Met, SEMA mutant or TK mutant in basal condition and upon HGF supplementation. Untransduced T47D were used as a control (CTRL). B) Wound healing Assay. Migration into the wound was quantified by time-lapse microscopy (one image every twenty minutes, for thirty-six hours), plotting the distance between the scratch margins calculated as mean of ten different measurements expressed as percentage of the initial distance. In TK mutant motility was double compared with wild type Met both in basal conditions (0,176 µm/sec vs 0,087 µm/sec) and upon ligand stimulation (0,250 µm/sec vs 0,100 µm/sec). Pictures represent WT Met and TK mutant at time 0 and after 15 hours. C) Invasion Assay. Each bar represents the relative ratio (per cent) of migrated cells in respect to wild type T47D, used as a control (CRTL). [(Met X /CTLR)* 100, where X= WT, SEMA mutant, TK mutant]. Experiments

  basal conditions and upon HGF stimulation. Error bars indicate standard deviations. * denotes statistically significant differences. D) Anchorage-independent Growth (Soft Agar) Assay. Colonies were scored after two weeks. Each bar represents the relative ratio (per cent) of number of colonies in respect to wild type T47D, used as a control (CRTL). [(Met X /CTLR)* 100, where X= WT, SEMA mutant, TK mutant]. Experiments were conducted in basal conditions and upon HGF stimulation. Error bars indicate standard deviations.

Figure 1 .Figure 2 .

 12 Figure 1. MET mutations in CUPs. Met receptor structure displaying mutations found in CUPs. § PM: plasma membrane. *P: sites of phosphorylation. Ƣ MRS: MET Related Sequence. • IPT: protein-protein interaction site 203x152mm (200 x 200 DPI)

  table are also represented. Supporting table 1. A summary on patients clinical data. ‡ wt denotes MET wild type status. ° n.a denotes data not available, patient withdrawn at follow up

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Acknowledgments

Supported by the Special Clinical Molecular Oncology Program of the Associazione Italiana per la Ricerca sul Cancro (AIRC); SB and AS are supported by individual grants from AIRC and (AS) Fondazione Cariparo.

We are grateful to Dr. Laura Casorzo for the helpful contribution in FISH analysis, to Dr. Antonella Balsamo for the assistance in the MSI analysis and to Dr. Carlo Zanon for the technical support in sequencing analysis; we thank Prof. Livio Trusolino for discussion and critical reading of the manuscript.


Ph.D Mr Ryszard Stolarski
email: stolarsk@biogeo.uw.edu.pl

Ph.D.; Katarzyna Ruszczyńska-Bartnik

Ph.D Maciej Maciejczyk

Ph.D Ryszard Stolarski

Keywords: eIF4E isoforms, Caenorhabditis elegans, mRNA 5' cap recognition, molecular dynamics eIF4E isoforms  Caenorhabditis elegans  mRNA 5'  Cap recognition  Molecular dynamics

Specific recognition and binding of the ribonucleic acid 5' termini (mRNA 5' cap) by the eukaryotic translation initiation factor 4E (eIF4E) is a key, rate limiting step in translation initiation. Contrary to mammalian and yeast eIF4Es that discriminate in favor of 7-methylguanosine cap, three out of five eIF4E isoforms from the nematode Caenorhabditis elegans as well as eIF4Es from the parasites Schistosome mansoni and Ascaris suum, exhibit dual binding specificity for both 7methylguanosine-and N2,N2,7-trimethylguanosine cap. To address the problem of the differences in the mechanism of the cap recognition by those highly homologic proteins, we carried out molecular dynamics simulations in water of three factors, IFE-3 and IFE-5 isoforms from C. elegans and murine eIF4E, in the apo form as well as in the complexes with 7-methyl-GDP and N2,N2,7-trimethyl-GDP. The results clearly pointed to a dynamical mechanism of discrimination between each type of the cap, viz. differences in mobility of the loops located at the entrance into the protein binding pockets during the cap association and dissociation. Additionally, our data showed that the hydrogen bond involving the N2-amino group of 7-methylguanosine and the carboxylate of glutamic acid was not stable. The dynamic mechanism proposed here differs from a typical, static one in that the differences in the protein-ligand binding specificity cannot be ascribed to formation and/or disruption of well defined stabilizing contacts.

Response to Reviewers:

Estimation of the binding free energies from common free molecular dynamics simulation is practically impossible. In theory they can be calculated from potential energies obtained from simulation, but in practice the convergence of the partition function is very poor (see Alen & Tildesley Computer Simulation of Liquids p. 213). There are known special techniques for this purpose (thermodynamic integration, thermodynamic perturbation) but their application for such a complex system is a very challenging problem. Our goal was to obtain general insight into dynamics of the system rather then computation of binding free energies.

With such a relatively well defined system it should also be possible to examine the role of specific side chains and study the effect of selected site point mutations on ligand binding energies and protein dynamics.

As the active site is highly conserved, it is difficult to pick single side-chain responsible for increased/decreased stability of a protein-ligand complex. Our simulations suggest that the discrimination between two types of cap is a consequence of different mobility of the loops in the eIF4E factors, which, in turn, might be a result of several point mutations and/or different lengths of those loops. The influence of several mutations on equilibrium constants is a difficult target for thermodynamic integration, because the precision of the method drops with increasing number of mutations. The problem might be addressed in a separate publication, although, as we wrote in page 4, the MD simulations of double mutated IFE-5 were already run (ref. [START_REF] Miyoshi | Discrimination between mono-and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E[END_REF]) but without success in explaining the molecular basis of the IFE-5 dual specificity vs. single specificity of IFE-3.

In general the paper is very descriptive and it it is difficult to extract any major insights or clearly answer any of the key questions. I feel the paper would be improved by some additional calculations and more detailed discussion.

Certainly, application of thermodynamic integration method would shed more light on the problem, although application of the TI method for such a large system is far from the routine task and requires a lot of computations. We think that it should be the subject of a separate publication.

Replies to Reviewer#2 comments:

1. "The only difference seems to be in the conformation of E90 carboxylate of S. mansoni eIF4E that is rotated by ~80deg in comparison to the orientation of the equivalent E103 [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF][START_REF] Marcotrigiano | Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP[END_REF] in murine eIF4E." A rotation of the carboxylate observed for the E103 side chain has also been observed in a glycerol bound structure of eIF4E see "Crystallization of eIF4E complexed with eIF4GI peptide and glycerol reveals distinct structural differences around the cap-binding site". Perhaps this should be referred to as it gives an indication of its inherent plasticity.

Yes, that is right. suitable comments and reference 32 were introduced in page 4 of Introduction and in page 8 (Results).

2. "This general view of the dynamic behaviour is confirmed by the experimental data derived for the apo form of human eIF4E by multidimensional NMR [START_REF] Boresch | Free energy simulations: the meaning of the individual contributions from a component analysis[END_REF] and for the murine factor by hydrogendeuterium exchange combined with electrospray mass spectrometry [START_REF] Volpon | Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands[END_REF]." Should reference ""Crystallization of eIF4E complexed with eIF4GI peptide and glycerol reveals distinct structural differences around the cap-binding site." as m7gtp is not present in one of the complexes in the asymetric unit and is also in broad agreement with the above statement.

Yes, that is right. An additional sentence and citation (ref. [START_REF] Brown | Crystallization of eIF4E complexed with eIF4GI peptide and grycerol reveals distinct structural differences around the cap-binding site[END_REF] was introduced in page 7.

3. "The structural differences in the regions of loops S1-S2, S3-S4, S5-S6, and S7-S8 (Fig. 2a),resulted in the formation of the positively charged pocket to anchor the cap phosphate chain (loops S1-S2 and S7-S8), followed by formation of the stacking triad and hydrogen bonds with the 7-methylguanine moiety via locking the W56 hinge (loop S1-S2) and rotating W102 (loop S3-S4) into the cap-binding site. Such a two-state model of the binding was previously proposed from fluorescence titration of murine eIF4E with structurally modified cap analogues and the parsing of the association free energy G [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF]." Does figure 2 Yes, that is right. Fig. 2 shows the dynamics of the apo factors and supports neither the two-step nor the one-step binding mechanism. We changed the suitable fragment to avoid ambiguity. Additionally, we referenced two our papers on stopped-flow experiments of the eIF4E-cap binding kinetics (refs. 51 and 52), and the paper of Slepenkov et al. (ref. 53), together with a short discussion in page 7 to address the discrepancy. 4: "and rotating W102 (loop S3-S4) into the cap-binding site" When discussing rotation of the W102 structure perhaps it would be prudent to reference crystal structures that show the ability of W102 to rotate ("Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives" and "Crystallization of eIF4E complexed with eIF4GI peptide and glycerol reveals distinct structural differences around the cap-binding site.") These papers show W102 in an open position and it ability to rotate into two alternative stacking positions with the methylated guanine base.

Yes that is right. An additional sentence and the references No. 32 and No. 50 were introduced in page 7.

5: "Binding of m7GDP to murine eIF4E results in bend of S7-S8 toward S5-S6 that helps to keep the ligand in the binding centre by water mediated interaction between K159 and S209." Ser209 is not present in most of the crystal structures in the PDB and I would assume as a result its role in any water mediated interactions would be minimal. It would be great to have the comment expanded to explain.

Yes, we agree. The interaction is only one of possible explanations for keeping the loops together, based on the analysis of eIF4E phosphorylated at Ser209 (refs. 55 and 25). We change the sentence to a more hypotetical one.

6: "analysis of the apo proteins provided some information on k+1 that reflects accessibility of the MMG-cap analogue and of the TMG-cap analogue for the binding sites of the three eIF4Es." Apo structures were generated by removing m7GDP. The apo NMR stucture is drastically quite different to this. Perhaps it should be emphasized that the simulated apo structures approximate a final conformation the ligands would dock into, especially if the two state model is correct which states that the triphosphates docks into the cap-binding site first. This is especially true if the large structural fluctuations observed in the NMR structure do not occur in the simulations.

Thank you for the remark. We added a sentence on this after the cited part in page 7.

Introduction

The 5' terminal structure of eukaryotic RNA polymerase II transcripts (RNA 5' cap) plays a crucial role in gene expression and regulation. The cap is specifically bound to several cellular and viral proteins, including various isoforms of eukaryotic translation factor eIF4E [START_REF] Joshi | Characterisation of mammalian eIF4E-famili members[END_REF], nuclear cap-binding complex CBC [START_REF] Lewis | The role of the cap structure in RNA processing and nuclear export[END_REF], DcpS scavenger enzyme [START_REF] Liu | The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphates[END_REF], poly(A) binding protein PABP [START_REF] Kühn | Structure and function of poly(A) binding proteins[END_REF], poly(A)-specific ribonuclease PARN [START_REF] Martinez | A 54-kDa fragment of the poly(A)-specific ribonuclease is oligomeric, processive, and capinteracting poly(A)-specific 3' exonuclease[END_REF], pokeweed antiviral protein PAP [START_REF] Hudak | Pokeweed antiviral protein binds to the cap structure of eukaryotic mRNA and depurinates the mRNA downstream of the cap[END_REF], cellular mRNA cap (guanine-N7) methyltransferase [START_REF] Fabrega | Structure and Mechanism of mRNA Cap (Guanine-N7) Methyltransferase[END_REF], human parneoplastic encephalomyeltis antigen HuD [START_REF] Wang | Structural basis for recognition of AU-rich element RNA by the HuD protein[END_REF], vaccinia virus 2'-O-methyltransferase VP39 [START_REF] Hodel | Specific protein recognition of an mRNA cap through its alkylated base[END_REF], influenza virus RNA polymerase [START_REF] Guilligay | The structural basis for cap binding by influenza virus polymerase subunit PB2[END_REF], and dimethyltransferase TGS1 [START_REF] Monecke | Structural basis for m 7 G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1[END_REF]. The eIF4E factors from vertebrates and yeast were shown to be highly selective for 7-methylguanosine cap (MMGcap) [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF], m 7 GpppN, N = G, A, U or C. In the nematodes Caenorhabditis elegans and Ascaris suum as well as in the parasitic flatworm Schistosoma mansoni a high population of messenger ribonucleic acids (mRNAs) contain a hypermethylated cap form, N 2 ,N 2 ,7trimethylguanosine cap (TMG-cap), m 3 2,2,7 GpppN, which is acquired along with a spliced leader during trans-splicing of pre-mRNA [START_REF] Blumenthal | Gene clusters and polycistronic transcription in eukaryotes[END_REF]. Affinity chromatography [START_REF] Jankowska-Anyszka | Multiple isoforms of eukaryotic protein syntesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono-and trimethylated mRNA cap structures[END_REF][START_REF] Keiper | Functional characterization of five eIF4E isoforms in Caenorhabditis elegans[END_REF], and fluorescence titration [START_REF] Stachelska | Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono-and trimethylated mRNA 5' cap analogues[END_REF][START_REF] Miyoshi | Discrimination between mono-and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E[END_REF] experiments showed that three out of five C. elegans eIF4E isoforms, IFE-1, IFE-2, IFE-5, are capable of binding specifically to the MMG-cap and to the TMG-cap. Two other isoforms, IFE-3, most similar to mammalian eIF4E, and IFE-4, related to the mammalian 4E-homologous protein 4E-HP, bind only to the MMG-cap. The dual binding specificity was also observed for eIF4Es from S. mansoni [START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF] and A. suum [START_REF] Lall | Contribution of trans-splicing, 5' -leader length, cap-poly(A) synergism, and initiation factors to nematode translation in an Ascaris suum embryo cell-free system[END_REF].

Values of the equilibrium dissociation constants, K d , for some selected complexes of the dual specificity eIF4Es with typical MMG-cap and TMG-cap analogues are shown in Table 1. The K d values for murine eIF4E are also shown for comparison. The values reported by various groups and derived by various titration methods, mainly fluorescence and isothermal calorimetry, can differ between each other. Nevertheless, the experimental data clearly show that both C. elegans IFE-3 and murine eIF4E strongly discriminate in favour of MMG-cap, ca. 11 kJ mol -1 and 16 kJ mol -1 , respectively, while in the case of C. elegans IFE-5 the binding energies are similar. The energy difference between S. mansoni eIF4E complexes is ca. 5 to 7 kJ mol -1 , depending on the titration experiment [START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF].

The TMG-cap occurs at the 5' terminus of small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and in telomerase RNA TLC1 [START_REF] Monecke | Structural basis for m 7 G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1[END_REF]. It is specifically recognized by Snurportin1 [START_REF] Strasser | Structural basis for m(3)G-capmediated nuclear import of spliceosomal UsnRNPs by snurportin1[END_REF], a receptor for spliceosomal small nuclear particles (snRNPs).

As shown by X-ray crystallography [START_REF] Hodel | Specific protein recognition of an mRNA cap through its alkylated base[END_REF][START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF][START_REF] Mazza | Large-scale induced fit recognition of an m 7 GpppG cap analogue by the human nuclear cap-binding complex[END_REF][START_REF] Calero | Structural basis of m(7)GpppG binding to the nuclear cap-binding protein complex[END_REF][START_REF] Marcotrigiano | Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP[END_REF][START_REF] Tomoo | Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)-and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region[END_REF][START_REF] Tomoo | Structural features of human initiation factor 4E, studied by X-ray crystal analysis,and molecular dynamics simulations[END_REF][START_REF] Gu | Insights into the structure, mechanism, and regulation of scavenger mRNA decapping activity[END_REF][START_REF] Chen | Crystal Structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping[END_REF][START_REF] Fechter | Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding[END_REF][START_REF] Af | The structure of the translation initiation factor eIF4E from wheat reveals a novel disulfide bond[END_REF] and multidimensional NMR [START_REF] Matsuo | Structure of translation factor eIF4E bound to m 7 GDP and interaction with 4Ebinding protein[END_REF] most of the cap-binding proteins converged at a common mechanism of the cap recognition via stacking of the 7-methylguanine moiety in between two aromatic amino acid side chains. The 7-methylguanine base possesses a net positive charge, which seems indispensable for its proper recognition, i.e. the 7-methylguanosine cannot be replaced by guanosine in the cap structure. In the snurportin1 complex with the dinucleotide TMG-cap analogue [START_REF] Strasser | Structural basis for m(3)G-capmediated nuclear import of spliceosomal UsnRNPs by snurportin1[END_REF] the sandwich stacking involves one tryptophan, and two bases of the cap, the first one, trimethylated, and the second one, unmethylated. In dimethyltransferase TGS1 the 7methylguanine moiety is stacked only on one tryptophan and a serine polar side chain limits the binding pocket on the opposite side [START_REF] Monecke | Structural basis for m 7 G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1[END_REF]. Except for the cation- stacking, the cap is also stabilized by a network of hydrogen bonds, direct or water mediated salt bridges, as well as less specific van der Waals and hydrophobic contacts. Only few exceptions have been found yet where the recognition specificity is entirely mediated through hydrogen bonds and van der Waals contacts with 7-methylguanine, i.e. cap methyltransferase [START_REF] Fabrega | Structure and Mechanism of mRNA Cap (Guanine-N7) Methyltransferase[END_REF], and reovirus polymerase 3 [START_REF] Tao | RNA Synthesis in a cage-structural studies of reovirus polymerase 3[END_REF].

In the mammalian, plant, and yeast eIF4E-cap complexes, two tryptophan aromatic residues take part in the sandwich cation- stacking with the 7-methylguanine moiety. Two hydrogen bonds involve N1H and N 2 H atoms of 7-methylguanosine and the carboxyl group of a conserved glutamic acid, and one hydrogen bond is observed between O 6 of m 7 G and the backbone amide nitrogen. Additional stabilizing interactions are between the phosphate chain of the cap and the arginine/lysine side chains of the protein. The first crystallographic structure of dual specificity eIF4E from Schistosoma mansoni in the complexes with two dinucleotide MMG-cap analogues [START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF] showed a similar binding mode as for the single specificity eIF4Es. The only difference seems to be in the conformation of E90 carboxylate of S. mansoni eIF4E that is rotated by ~80 in comparison to the orientation of the equivalent E103 [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF][START_REF] Marcotrigiano | Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP[END_REF] in murine eIF4E. This precludes the formation of two strong hydrogen bonds with the 7-methylguanine moiety. A similar rotation was also observed in the human eIF4E ternary complex with eIF4GI peptide and a glycerol molecule located in the cap-binding centre [START_REF] Brown | Crystallization of eIF4E complexed with eIF4GI peptide and grycerol reveals distinct structural differences around the cap-binding site[END_REF]. Still, the contribution of that conformational change to MMG-cap vs. TMG-cap binding specificity remains unclear. The NMR analysis of the MMG-cap and TMG-cap complexes with S. mansoni eIF4E [START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF] showed substantial chemical shift perturbation for ca.

15 amino acids, most of them distributed around the cap-binding pocket. Based on the crystallographic and NMR data the authors suggested that intrinsic and specific conformational flexibility of the S. mansoni eIF4E plays a crucial role in the TMG-cap binding, analogous to an "induced fit" mechanism. On the contrary, combined mutagenesis studies and molecular dynamics simulations of C. elegans dual specificity IFE-5 led to a "structural" rather than a "dynamic" model. Larger width and depth of the cap-binding pocket was postulated to be responsible for the TMG-cap binding specificity [START_REF] Miyoshi | Discrimination between mono-and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E[END_REF]. Replacement of two amino acids, N64Y/V65L decreased the size of the pocket and gave rise to discrimination against TMG-cap by steric hindrance. However, it was noted that dual specific A. suum eIF4E does contain Y64 and L65 residues [START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF]. Unfortunately, discrimination between TMG-cap and MMG-cap by snurportin1 is based on a mechanism that differs from that expected for the eIF4E factors [START_REF] Strasser | Structural basis for m(3)G-capmediated nuclear import of spliceosomal UsnRNPs by snurportin1[END_REF][START_REF] Goette | Molecular determinants of snurportin 1 ligand afinity and structural response upon binding[END_REF].

In order to get an insight into the mechanism of dual specificity in the cap recognition by some of highly homologic eIF4Es, we performed long-lasting molecular dynamics (MD) simulations in water for three selected eIF4E homologues, murine eIF4E as well as C. elegans IFE-3 and IFE-5, each of them in the apo form and in the complexes with m 7 GDP or with m 3 2,2,7 GDP (Scheme 1). The results point to a dynamic mechanism of discrimination between the mono-and hypermethylated cap structures.

Theory and methods

Initial setup

The starting structure of the complex of truncated murine eIF4E(28-217) with m 7 GDP for molecular dynamics simulations was taken from crystallography (PDB code: 1EJ1; [START_REF] Marcotrigiano | Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP[END_REF]). The missing atoms of some of the amino acid side chains were completed by SCWRL [START_REF] Brower | Prediction of protein side-chains rotamers from backbone dependent rotamer library: a new homology modeling tool[END_REF]. The hydrogen atoms were added in Insight II (Accelrys Software Inc., U.S.A.). The starting structures of IFE isoforms were obtained by homology modeling with murine eIF4E bound to m 7 GDP as a template, with 51% and 42% sequence identity to IFE-3 and IFE-5, respectively. The multiple sequence alignment was performed by CLUSTAL W [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choise[END_REF]. Ten structures for each isoform were obtained using the program MODELLER [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF]. Additional harmonic constraints were introduced for the distances between the protein and the cap atoms that were engaged in hydrogen bonds, salt bridges and van der Waals contacts. Subsequently, the resulting structures were subjected to detailed analysis regarding packing of the residues, steric hindrance, and loop conformations. Based on the analysis one representative structure was chosen for each isoform. Due to high sequence homology the modeled IFE structures were very similar to that of the eIF4E template (Fig. 1), especially regarding the main polypeptide chains. The structures of the complexes of three 4E factors with m 3 2,2,7 GDP, and of murine eIF4E with GDP, were constructed by adding two methyl groups at N 2 and by removing the methyl group from N7 in the protein-m 7 GDP complexes, respectively. The apo proteins were obtained by removing m 7 GDP from the complexes.

The ESP charges of the isolated ligands were calculated at HF/6-31G(d,p) level using Gaussian 94 (Gaussian Inc., Pittsburg PA, U.S.A.)

Molecular dynamics simulation and analysis

The MD simulations were carried out by the program Sigma [START_REF] Mann | Sigma MD program and a generic interface applicable to multifunctional programs with complex, hierarchical command structure[END_REF] using CHARMM22 force field [START_REF] Kerell | All-atom empirical potential for molecular modeling and dynamics studies of ptoteins[END_REF]. Each protein or complex was subjected to energy minimization without electrostatic interaction and immersed in an equilibrated TIP3P water box [START_REF] Jorgensen | Comparison of simple potential function for simulating liquid water[END_REF] keeping at least 10Ǻ shell thickness from the protein surface. The simulation procedure consisted of several equilibration MD runs preceded and followed by 500-step energy minimization , and a subsequent regular MD run, as follows. First, energy minimization and 48 ps dynamics of water molecules was performed keeping the protein or the complex immobilized. Second, energy minimization of the protein or of the complex with the water molecules kept fixed was followed by stepwise heating of the whole system from 50K to 300K for 82.56 ps. The initial velocities at each temperature were taken from Maxwell-Boltzmann distribution. The equilibration MD runs were performed in the nVT ensemble and the regular MD simulations were performed in the npT ensemble [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF] at temperature T = 300K and pressure p = 1 atm.

The SHAKE algorithm [START_REF] Ryckaert | Numerical integration of the Cartesian equations of mosion of a system with constraints: molecular dynamics of n-alkanes[END_REF] was applied to constrain the bonds. The electrostatic interactions were calculated by multiple time step [START_REF] Tuckreman | Reversible multiple time scale molecular dynamics[END_REF] with a double cut-off at 6Å and 10 Å. Short-, middle-, and long range interactions, according to particle-mesh Ewald method [START_REF] Darden | Particle mesh Ewald: an Nlog(N) method for Ewald sums in large systems[END_REF][START_REF] Schlick | Algorithmic challanges in computational molecular biophysics[END_REF], were calculated for an integration time-step of 2, 4, and 12 fs, respectively. The simulations were run for 5 ns in the case of the apo proteins or for 10 ns in the case of the complexes, in order to reach at least partial equilibrium according to a stability criterion for the fluctuations of root-mean-square-deviation (RMSD) of the proteins' C  atoms.

The conformations of the solute on a simulated trajectory were written down every 0.96 ps and analyzed regarding interatomic distances and torsion angles. Essential dynamics (ED) analysis of selected, equilibrium parts of the MD trajectories was performed according to Amadei et al. [START_REF] Amadei | Essential dynamics of proteins[END_REF].

Results

An experimentally observed equilibrium association constant K as expressed in terms of the molar concentrations of the reactants in a protein-ligand association is related to the standard Gibbs free energy Gof the association process at temperature T, G= RTlnK as . Hence K as is a quantitative measure of the ligand affinity for the protein. Comparison of the K as values for a series of structurally modified cap analogues enabled parsing of G into separate contributions from various stabilizing contacts inside the eIF4E cap-binding pocket [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF].

Bearing in mind an approximate character of the approach due to lack of additivity of the entropic terms [START_REF] Mark | Decomposition of the free energy of a system in terms of specific interactions. Implications fot theoretical and experimental studies[END_REF][START_REF] Boresch | Free energy simulations: the meaning of the individual contributions from a component analysis[END_REF], combination of the crystallographic structure with such G analysis provided molecular mechanism of specific binding between the cap and eIF4E [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF]. However, applying the procedure to detect the discrimination mechanism between MMG-and TMG-cap by some eIF4Es [START_REF] Miyoshi | Discrimination between mono-and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E[END_REF][START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF] has failed. The structures of IFE isoforms derived by homology modeling were very similar to that of the eIF4E template due to high sequence homology (Fig. 1). Therefore, the structural differences of potential importance for the MMG-vs. TMGcap binding selectivity have not been identified. This prompted us to evaluate a discrimination mechanism of a dynamic type. The equilibrium association constant, K as = k +1 /k -1 , depends on the ligand ability to form and leave the complex, expressed by kinetic rate constants k +1 and k - 1 , respectively. Higher k +1 and/or lower k -1 values give rise to an increase of K as . Since it was impossible to calculate theoretically the rate constants from all-atom MD simulations, we assumed that the MD analysis of the apo proteins provided some information on k +1 that reflects accessibility of the MMG-cap analogue and of the TMG-cap analogue for the binding sites of the three eIF4Es. Similarly, the MD analysis of the three factors, each bound to either MMG-cap or TMG-cap, might provide some hints on the stability of the complexes that influences their dissociation kinetic constants k -1 . It should be emphasized that the simulated apo structures were generated by removing the ligand, and therefore they differ from those derived experimentally, at least in the case of the NMR structure of murine apo-eIF4E [START_REF] Volpon | Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands[END_REF].

The simulated apo structures may be regarded as those, which approximate final conformations the ligands would dock into, especially if the cap phosphate chain anchores first as postulated by a two-state model of the cap-binding [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF] (see below). It is also worth to note, that the large structural fluctuations observed in the NMR structure of apo-eIF4E do not occur during our MD simulations.

The MD trajectories of the apo eIF4Es display enhanced flexibility of the loops around the entrance to the cap-binding pocket (Fig. 2), especially S1-S2 and S7-S8 loops, while the secondary structure elements remained unchanged. This general view of the dynamic behavior is confirmed by the experimental data derived for the apo form of human eIF4E by multidimensional NMR [START_REF] Volpon | Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands[END_REF], for the cap-free human eIF4E in the complex with glycerol that seems to represent an intermediate state between the apo and the cap-bound form [START_REF] Brown | Crystallization of eIF4E complexed with eIF4GI peptide and grycerol reveals distinct structural differences around the cap-binding site[END_REF], and for the murine factor by hydrogen-deuterium exchange combined with electrospray mass spectrometry [START_REF] Rutkowska-Wlodarczyk | Structural changes of eIF4E upon binding to the mRNA 5' monomethylguanosine and trimethylguanosine cap[END_REF]. The secondary elements were preserved in apo eIF4E while the loops exhibited mobility on the ns -ps time scale that became abrogated upon the cap binding. The structural differences in the regions of loops S1-S2, S3-S4, S5-S6, and S7-S8 (Fig. 2a) resulted in the formation of the positively charged pocket to anchor the cap phosphate chain (loops S1-S2 and S7-S8), and the formation of the stacking triad and hydrogen bonding with the 7-methylguanine moiety via locking the W56 hinge (loop S1-S2) and rotating W102 (loop S3-S4) into the cap-binding site. The ability of W102 containing loop S3-S4 to rotate from the open (apo) state into the closed form was confirmed by both crystallography [START_REF] Brown | Crystallization of eIF4E complexed with eIF4GI peptide and grycerol reveals distinct structural differences around the cap-binding site[END_REF] and NMR studies [START_REF] Volpon | Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands[END_REF]. Moreover, an alternative orientation of W102 indole ring was found by the diffraction studies of the eIF4E complexes with 7-benzyl-GMP and 7-(p-fluorobenzyl)-GMP.

The W102 residue flips through 180 [START_REF] Brown | Crystallographic and mass spectrometric characterisation of eIF4E with N 7 -alkylated cap derivatives[END_REF] in comparison with those complexes which contain the 7-methylguanosine caps [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF][START_REF] Marcotrigiano | Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP[END_REF][START_REF] Tomoo | Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)-and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region[END_REF]. All those observations agree with the a two-state model of the cap-binding [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF], in which anchoring of the phosphate chain was followed by a cooperative formation of the stacking triad and hydrogen bonds. This model, derived from analysis of the fluorescence titration of eIF4E with structurally modified cap analogues , was confirmed by kinetic stopped flow experiments [START_REF] Blachut-Okrasinska | Stopped-flow and Brownian dynamic studies of electrostatic effects in the kinetics of binding of 7-methyl-GpppG to the protein eIF4E[END_REF][START_REF] Niedzwiecka | Biophysical approach to studies of cap-eIF4E interaction by synthetic cap analogs[END_REF]. Contrary to our analysis, Slepenkov et al. [START_REF] Slepenkov | Stopped-flow kinetic analysis of eIF4E and phosphorylated eIF4E binding to cap analogs and capped oligoribonucleotides[END_REF] performed and analyzed the stopped flow experiments differently, and postulated a one-step mechanism However, our results are confirmed not only by the standard analysis of the stop-flowed kinetic traces under pseudo first-order conditions [START_REF] Blachut-Okrasinska | Stopped-flow and Brownian dynamic studies of electrostatic effects in the kinetics of binding of 7-methyl-GpppG to the protein eIF4E[END_REF] but also by running the experiments under the second-order conditions combined with numerical integration of the suitable differential kinetic equations [START_REF] Niedzwiecka | Biophysical approach to studies of cap-eIF4E interaction by synthetic cap analogs[END_REF] by the state-of-the-art program DynaFit [START_REF] Kuzmic | Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV protease[END_REF].

The flexibility of the loops seem to be crucial for the discrimination between m 7 GDP and m 3 2,2,7 GDP by murine eIF4E and C. elegans IFE-3 and IFE-5. The calculated distances between S7-S8 and S5-S6, and between S5-S6 and S1-S2, on the final parts of the MD trajectories (Fig. 2b) are ca. 10Å greater for IFE-5 that binds the TMG-cap than for IFE-3 and murine eIF4E that are specific for the MMG-cap only. Hence, the TMG-analogue with two additional methyl groups can easy penetrate the IFE-5 binding pocket contrary to other factors.

A similar analysis of the dynamics of the protein-cap complexes shows stable contacts between the cap phosphates and the arginine/lysine side chains, irrespective of the bound analogue. This is consistent with the anchoring character of the phosphate groups in the cap stabilization inside the eIF4E binding slot [START_REF] Niedzwiecka | Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins[END_REF]. On the contrary, the mutual orientations of the rings in the cation- stacking triad undergo larger fluctuations in respect to the starting structure. As expected, the largest changes are observed in the eIF4E-GDP complex that is stabilized by weaker - stacking, i.e. a perpendicular orientation of the W56 and G rings and a shift of W102 deeper into the binding pocket. In the case of all the other complexes the stacking triad is kept principally unchanged, with relatively larger fluctuations of W102. A temporary increase of the distance between W102 and m 7 G rings are observed in the eIF4Em 3 2,2,7 GDP and IFE-3 -m 3 2,2,7 GDP, but not in IFE-5-m 3 2,2,7 GDP, complexes. On the other hand, the hydrogen bond between the N 2 -amino group of m 7 G and the E103 carboxylate (Fig. 3) is being broken and reformed due to shifts of the E103 side chain into the bulk solvent.

Inherent "plasticity" of that part of the cap-binding centre, resulting in a rotation of E103 side chain and a movement of W102 side chain, was also observed in the cap-free eIF4E ternary complex with eIF4GI peptide and glycerol in the cap-binding pocket [START_REF] Brown | Crystallization of eIF4E complexed with eIF4GI peptide and grycerol reveals distinct structural differences around the cap-binding site[END_REF]. Therefore, the low affinity of the TMG-cap for murine eIF4E cannot be explained by lack of that hydrogen bond due to the hypermethylation.

The distance between loops S7-S8 and S5-S6 in the eIF4E-cap complexes (Fig. 4) is shown to correlate with the affinity of various cap types for the eIF4E isoforms. Binding of m 7 GDP to murine eIF4E results in bend of S7-S8 toward S5-S6 that might help to keep the ligand in the binding centre by water mediated interaction between K159 and e.g. S209. As shown previously [START_REF] Zuberek | Influence of electric charge variation at residues 209 and 159 on the interaction of eIF4E with the mRNA 5' terminus[END_REF] K159 is very important for binding the capped mRNA. Closing the entrance to the cap-binding pocket by decreasing of the distance between S209 and K159 was also observed in MD simulations of phosphorylated eIF4E [START_REF] Tomoo | Structural features of human initiation factor 4E, studied by X-ray crystal analysis,and molecular dynamics simulations[END_REF]. In the complexes with the low affinity ligands, m 3 2,2,7 GDP and GDP, the distance between the loops is larger, thus making it easier for those ligands to leave the complexes. Similarly, the distance between the loops in the IFE-3 complex with m 7 GDP is much smaller than in the IFE-3 complex with m 3 2,2,7 GDP, while for both IFE-5 complexes it is kept fairly large, irrespective of the ligand. The analysis of the overall dynamics of the cap-bound eIF4E isoforms was also carried out by ED analysis of the covariance matrix of the atomic displacements [START_REF] Amadei | Essential dynamics of proteins[END_REF]. The scalar products of the the vectors representing the normalized C  displacements and the eigenvectors corresponding to the largest eigenvalue ( = 1) show non-Gaussian distributions with 2 -3 maxima. This can be interpreted as correlated, long-range movements, in which the loops oscillate around several mean positions (Fig. 5). The histograms for the scalar products of the eigenvectors corresponding to lower eigenvalues ( = 10) are Gaussian, as expected for equilibrated, independent and harmonic motions.

Discussion

The The presence of two methyl substituents in the amino group of 7-methylguanosine brakes at least one stabilizing hydrogen bond in the cap-binding protein pockets and may lead to a substantial decrease of the association constants observed for the complexes with TMG-cap.

On the other hand, the dual specificity cap-binding proteins possess high homology and structure similarity to those that discriminate between the MMG-cap and the TMG-cap.

Hence, it is a great challenge to conceive a molecular model of the discrimination vs. dual specificity for the protein-cap association. The explanations usually do not go beyond formulations like "the differences in the size of the cap-binding pocket in the C. elegans isoforms of eIF4E" [START_REF] Strasser | Structural basis for m(3)G-capmediated nuclear import of spliceosomal UsnRNPs by snurportin1[END_REF]. Our approach to elucidate the specificity of the caps recognition can be expressed in terms of the ligand ability to enter or leave the apo protein binding pocket, since the equilibrium association constant is determined by the ratio of the two rate constants.

Although we were not able to calculate the values of the rate constants by all-atom MD simulations, the comparative analysis of the MD trajectories of the apo-and cap-bound factors provides a more detailed explanation for the differences in the binding specificity of two C. elegans eIF4E isoforms, IFE-3 and IFE-5, than those published hitherto. The dynamic mechanism of the discrimination between two types of the cap may be ascribed to differences in mobility of the loops around the entrance to the protein cap-binding pockets, especially S7-S8 loop. Our results show also higher rigidity of the cation- stacking triad and of the stabilizing interactions (salt bridges, hydrogen bonds) involving the cap phosphate chain compared with a more flexible character of the hydrogen bonds. The results of our computer modeling are generally consistent with the experimental, structural and dynamic, data [START_REF] Brown | Crystallization of eIF4E complexed with eIF4GI peptide and grycerol reveals distinct structural differences around the cap-binding site[END_REF][START_REF] Volpon | Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands[END_REF][START_REF] Rutkowska-Wlodarczyk | Structural changes of eIF4E upon binding to the mRNA 5' monomethylguanosine and trimethylguanosine cap[END_REF].

Conclusions

Discrimination between two types of the cap, MMG-cap and TMG-cap, consist neither in the differences in the stacking energy nor in well-defined structural differences inside the capbinding pocket. Both 7-methylguanosine and its hypermethylated form were found to stack equally well in between two amino acid aromatic rings [START_REF] Worch | Stacking efficienct and flexibility analysis of aromatic amino acids in cap-binding proteins[END_REF], and the structure of S. mansoni eIF4E in complex with the MMG-cap analogues [START_REF] Liu | Structural insight into parasite eIF4E binding specificity for m 7 G and m 2,2,7 G mRNA cap[END_REF], showed a very similar mode of binding to that of the single specificity eIF4Es. MD simulations based on the known structures of the cap-eIF4E complexes provided means to evaluate the discrimination mechanism. Contrary to the comparative analysis of the "static" net of stabilizing contacts inside the cap-binding pockets of highly homologic eIF4Es, we took into account the differences in the dynamics of the formation and dissociation of the eIF4E-cap complexes. An exact specification of the role of particular amino acids in the proposed dynamical mechanism, e.g. their mutual interactions and/or their interactions with various cap structures, needs further investigations.

  really support a two step model. Also if it does why does it not agree with single step binding mechanism proposed by Slepenkov et al. (Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides EVIDENCE FOR A ONE-STEP BINDING MECHANISM*) Perhaps this should also be referenced.

  knowledge of the molecular basis of the RNA 5' cap structure recognition by the capbinding proteins is prerequisite for understanding possible mechanisms of the cap functioning in various types of the gene expression processes in eukaryotes, such as translation initiation, mRNA splicing, and export of RNAs to the cytoplasm. It seems that various evolutionary unrelated cap-binding proteins converged on a similar general mechanism of the cap recognition. Subtle modifications of the general recognition mechanism of the cap may lead to differences in the protein functions, e.g. the diverse role of two aromatic amino acids that stack with the 7-methylguanosine moiety[START_REF] Hodel | Specific protein recognition of an mRNA cap through its alkylated base[END_REF][START_REF] Fechter | Recognition of mRNA cap structures by viral and cellular proteins[END_REF][START_REF] Worch | Diverse role of three tyrosines in binding of the RNA 5' cap to the human nuclear cap binding complex[END_REF]. The methyl group at N7 in the cap structure imparts a net positive charge to the guanine ring, and results in more efficient stacking compared with the unmethylated base. Quantum mechanical calculations showed that a typical energy of the cation- stacking of the m 7 G base in the complex with the tyrosine or tryptophan aromatic ring is in a range -11.4 kcal mol -1 to -16.23 kcal mol -1 , while the G/Y - stacking energy is ca. -6 kcal mol -1[START_REF] Worch | Stacking efficienct and flexibility analysis of aromatic amino acids in cap-binding proteins[END_REF]. Additional methyl groups at N 2 do not change the stacking ability of the cap. The stacking energy of m 3 2,2,7 G/W276 in Snurportin 1, -12.52 kcal mol -1 , is close to typical values obtained for the m 7 G/W complexes.

Figure captions Scheme 1 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

 112345 Figure captions

Acknowledgments

This work was supported by the Polish Ministry of Science and Higher Education, grant No.

N N301 035 936. The MD simulations were performed in the Interdisciplinary Centre for Mathematical and Computational Modelling (ICM) at the University of Warsaw. We are indebted to Dr. Joanna Trylska for critical reading of the manuscript.

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