Unveiling mutation effects on the structural dynamics of the main protease from SARS-CoV-2 with hybrid simulation methods.

The main protease of SARS-CoV-2 (called Mpro or 3CLpro) is essential for processing polyproteins encoded by viral RNA. Several Mpro mutations were found in SARS-CoV-2 variants, which are related to higher transmissibility, pathogenicity, and resistance to neutralization antibodies. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. In this study, we used a hybrid simulation method to generate intermediate structures along the six lowest frequency normal modes and sample the conformational space and characterize the structural dynamics and global motions of WT SARS-CoV-2 Mpro and 48 mutations, including mutations found in P.1, B.1.1.7, B.1.351, B.1.525 and B.1.429+B.1.427 variants. We tried to contribute to the elucidation of the effects of mutation in the structural dynamics of SARS-CoV-2 Mpro. A machine learning analysis was performed following the investigation regarding the influence of the K90R, P99L, P108S, and N151D mutations on the dimeric interface assembling of the SARS-CoV-2 Mpro. The parameters allowed the selection of potential structurally stable dimers, which demonstrated that some single surface aa substitutions not located at the dimeric interface (K90R, P99L, P108S, and N151D) are able to induce significant quaternary changes. Furthermore, our results demonstrated, by a Quantum Mechanics method, the influence of SARS-CoV-2 Mpro mutations on the catalytic mechanism, confirming that only one of the chains of the WT and mutant SARS-CoV-2 Mpros are prone to cleave substrates. Finally, it was also possible to identify the aa residue F140 as an important factor related to the increasing enzymatic reactivity of a significant number of SARS-CoV-2 Mpro conformations generated by the normal modes-based simulations.

Effect of phosphorylation on the structural dynamics, thermal stability of human dopamine transporter: A simulation study using normal modes, molecular dynamics and Markov State Model.

The Human Dopamine Transporter (hDAT) plays an essential role in modulating the Influx/Efflux of dopamine, and it is involved in the mechanism of certain neurodegenerative diseases such as Parkinson's disease. Several studies have reported important states for Dopamine transport: outward-facing open state (OFo), the outward-facing closed state (OFc), the holo-occluded state closed (holo), and the inward-facing open state (IFo). Furthermore, experimental assays have shown that different phosphorylation conditions in hDAT can affect the rate of dopamine absorption. We present a protocol using hybrid simulation methods to study the conformational dynamics and stability of states of hDAT under different phosphorylation sites. With this protocol, we explored the conformational space of hDAT, identified the states, and evaluated the free energy differences and the transition probabilities between them in each of the phosphorylation cases. We also presented the conformational changes and correlated them with those described in the literature. There is a thesis/hypothesis that the phosphorylation condition corresponding to NP-333 system (where all sites Ser/Thr from residue 2 to 62 and 254 to 613 are phosphorylated, except residue 333) would decrease the rate of dopamine transport from the extracellular medium to the intracellular medium by hDAT as previously described in the literature by Lin et al., 2003. Our results corroborated this thesis/hypothesis and the data reported. It is probably due to the affectation/changes/alteration of the conformational dynamics of this system that makes the intermediate states more likely and makes it difficult to initial states associated with the uptake of dopamine in the extracellular medium, corroborating the experimental results. Furthermore, our results showed that just single phosphorylation/dephosphorylation could alter intrinsic protein motions affecting the sampling of one or more states necessary for dopamine transport. In this sense, the modification of phosphorylation influences protein movements and conformational preferences, affecting the stability of states and the transition between them and, therefore, the transport.

ABCG2/BCRP transport mechanism revealed through kinetically excited targeted molecular dynamics simulations.

ABCG2/BCRP is an ABC transporter that plays an important role in tissue protection by exporting endogenous substrates and xenobiotics. ABCG2 is of major interest due to its involvement in multidrug resistance (MDR), and understanding its complex efflux mechanism is essential to preventing MDR and drug-drug interactions (DDI). ABCG2 export is characterized by two major conformational transitions between inward- and outward-facing states, the structures of which have been resolved. Yet, the entire transport cycle has not been characterized to date. Our study bridges the gap between the two extreme conformations by studying connecting pathways. We developed an innovative approach to enhance molecular dynamics simulations, 'kinetically excited targeted molecular dynamics', and successfully simulated the transitions between inward- and outward-facing states in both directions and the transport of the endogenous substrate estrone 3-sulfate. We discovered an additional pocket between the two substrate-binding cavities and found that the presence of the substrate in the first cavity is essential to couple the movements between the nucleotide-binding and transmembrane domains. Our study shed new light on the complex efflux mechanism, and we provided transition pathways that can help to identify novel substrates and inhibitors of ABCG2 and probe new drug candidates for MDR and DDI.

Water dynamics in rigid ionomer networks.

The dynamics of water within ionic polymer networks formed by sulfonated poly(phenylene) (SPP), as revealed by quasi-elastic neutron scattering (QENS), is presented. These polymers are distinguished from other ionic macromolecules by their rigidity and therefore in their network structure. QENS measurements as a function of temperature as the fraction of ionic groups and humidity were varied have shown that the polymer molecules are immobile while absorbed water molecules remain dynamic. The water molecules occupy multiple sites, either bound or loosely constrained, and bounce between the two. With increasing temperature and hydration levels, the system becomes more dynamic. Water molecules remain mobile even at subzero temperatures, illustrating the applicability of the SPP membrane for selective transport over a broad temperature range.

Treatment and outcomes for patients with depression who are partial responders to SSRI treatment: post-hoc analysis findings from the FINDER European observational study.

BACKGROUND: Remission is the goal in depression, but in practice many patients only experience a partial response to treatment. We sought to determine the prevalence, management and subsequent outcomes of partial responder patients. METHODS: Patients enrolled in the naturalistic Factors Influencing Depression Endpoints Research (FINDER) study with the Hospital Anxiety and Depression Scale depression subscale (HADS-D) score >10 at baseline who received only SSRI(s) between 0 and 3 months comprised the study cohort (n=1147). Patients were categorized as remitters, partial responders or non-responders at 3 months and then followed up at 6 months. RESULTS: At 3 months, 29.4% of the study population were considered non-responders, 27.6% were partial responders, and 39.3% were remitters. Most partial responders at 3 months remained on the same SSRI for the next 3 months. Of the 247 partial responders at 3 months and remained on the same SSRI(s) between 3 and 6 months, 10.9% met criteria for non-response at 6 months, 32.4% remained partial responders, and 56.3% achieved remission. Quality of life outcomes for the partial responders were significantly worse than those in remission (p<0.05). LIMITATIONS: FINDER was an observational study; the current analysis was conducted post-hoc. Multivariable methods were not applied and findings are primarily descriptive and exploratory. CONCLUSIONS: Partial response is common and patients in partial response have a poorer quality of life than those achieving remission. Despite this, the majority of partial responders continue to take the same SSRI. Our findings underscore the importance of continuing to strive for remission.

Duloxetine 60 mg/day for the prevention of depressive recurrences: post hoc analyses from a recurrence prevention study.

OBJECTIVE: To assess the efficacy of duloxetine 60 mg/day in the prevention of depressive recurrence in patients with major depressive disorder (MDD). METHODS: Patients having at least three episodes of MDD in the past 5 years received open-label (OL) duloxetine 60-120 mg/day for up to 34 weeks. Patients meeting response criteria were then randomised to either duloxetine or placebo for up to 52 weeks of double-blind maintenance treatment. Only patients taking duloxetine 60 mg/day during the OL phase, and randomised to either duloxetine (remained on 60 mg/day dose) or placebo, were included in this post hoc analysis. The primary outcome measure was time to recurrence of a major depressive episode. The 17-item Hamilton Rating Scale for Depression (HAMD(17)) was used to evaluate depressive symptomatology. Global and physical functioning and pain were also assessed. Safety and tolerability were assessed via analysis of treatment-emergent adverse events (TEAEs), vital signs and weight. RESULTS: A total of 124 patients were randomised to duloxetine 60 mg/day (n = 64) or placebo (n = 60). Time to depressive recurrence was significantly longer in duloxetine-treated patients compared with placebo-treated patients (p = 0.001). During the double-blind maintenance phase, 31.7% of placebo-treated patients experienced a depressive recurrence compared with 12.5% of duloxetine-treated patients (p = 0.004). The HAMD(17) total score and most of its subscales as well as the Clinical Global Impression of Severity (CGI-S), significantly worsened in the placebo group compared with the duloxetine 60 mg/day group. There were no significant differences between treatment groups in TEAEs, discontinuations because of adverse events, vital signs or weight. CONCLUSIONS: Treatment with duloxetine 60 mg/day was associated with a longer time to depressive recurrence and a significantly lower recurrence rate compared with placebo.

Presence and predictors of pain in depression: results from the FINDER study.

BACKGROUND: Patients with depression often experience pain. There is limited understanding of the relation between pain and other symptoms (depressive, anxious and non-painful somatic symptoms). This exploratory study assesses pain severity and interference of pain with functioning in a clinically depressed population and investigates the relation between the different groups of symptoms. METHODS: FINDER was a 6-month prospective, observational study investigating health-related quality of life of outpatients with depression initiating antidepressant treatment. Patients completed ratings on the Hospital Anxiety and Depression Scale (HADS), Somatic Symptom Inventory (SSI-28), and overall pain severity and interference of pain with functioning using Visual Analogue Scales (VAS) at baseline and at 3 and 6 months. Regression analyses identified factors associated with overall pain severity and interference of pain with functioning, at baseline and over the observation period. RESULTS: Of 3468 eligible patients at baseline, 56.3% experienced moderate to severe pain and 53.6% had moderate to severe pain-related interference with functioning. At 6 months of follow-up, these proportions decreased to 32.5% and 28.1%, respectively. Higher baseline SSI-somatic scores (non-painful) were strongly associated with greater pain severity and greater pain-related interference with functioning at baseline and over 6 months. Certain socio-demographic (increasing age, being unemployed) and depression-related factors (more previous episodes, longer duration of current episode) were also significantly associated with greater pain severity and interference over 6 months, while higher baseline severity of depression (HADS-D) and further education were associated with less severe pain or pain-related interference with functioning over 6 months. CONCLUSIONS: Over half of depressed patients in this study experienced moderate to severe pain. Painful somatic symptoms appear to be closely related to non-painful somatic symptoms, more than to depressive or anxious symptoms suggesting that painful and non-painful somatic symptoms can be considered as one group of 'somatic symptoms,' all of them associated with depressive and anxious symptoms.

Duloxetine in the treatment of major depressive disorder: a placebo- and paroxetine-controlled trial.

OBJECTIVE: Duloxetine doses of 80 and 120 mg/day were assessed for efficacy and safety in the treatment of major depressive disorder (MDD). METHODS: In this randomized, double-blind trial, patients age > or =18 meeting DSM-IV criteria for MDD were randomized to placebo (N=99), duloxetine 80 mg/day (N=93), duloxetine 120 mg/day (N=103), or paroxetine 20 mg/day (N=97). The primary outcome measure was mean change from baseline in the 17-item Hamilton rating scale for depression (HAMD(17)) total score after 8 weeks of treatment; a number of secondary efficacy measures also were assessed. Safety and tolerability were assessed via collection and analysis of treatment-emergent adverse events (TEAEs), vital signs, and weight. The Arizona sexual experiences scale was used to assess sexual functioning. Patients who had a > or =30% reduction from baseline in the HAMD(17) total score at the end of the acute phase entered a 6-month continuation phase where they remained on the same treatment as they had taken during the acute phase; efficacy and safety/tolerability outcomes were assessed during continuation treatment. RESULTS: More than 87% of patients completed the acute phase in each treatment group. Duloxetine-treated patients (both doses) showed significantly greater improvement (P<0.05) in the HAMD(17) total score at week 8 compared with placebo. Paroxetine was not significantly different from placebo (P=0.089) on mean change on the HAMD(17). Duloxetine 120 mg/day also showed significant improvement on most secondary efficacy measures (six of nine) compared with placebo while duloxetine 80 mg/day (three of nine) and paroxetine (three of nine) were significantly superior to placebo on fewer secondary measures. HAMD(17) mean change data from this study and an identical sister study were pooled as defined a priori for the purposes of performing a non-inferiority test versus paroxetine. Both duloxetine doses met statistical criteria for non-inferiority to paroxetine. TEAE reporting rates were low in all treatment groups and no deaths occurred in the acute or continuation phases. CONCLUSIONS: The efficacy of duloxetine at doses of 80 and 120 mg/day in the treatment of MDD was demonstrated. Tolerability, as measured by TEAEs, and safety were similar to paroxetine 20 mg/day and consistent with previous published data on duloxetine in the treatment of MDD.

Duloxetine 60 mg once daily in the treatment of milder major depressive disorder.

There is ongoing debate regarding the effectiveness of antidepressants in patients with milder major depressive disorder (MDD). This post-hoc analysis evaluated the efficacy and tolerability of duloxetine in the subset of 159 (75 duloxetine and 84 placebo) patients with milder MDD (baseline HAMD17 total score > or = 15 and < or = 18) who were treated once daily with duloxetine 60 mg or placebo in two identical, 9-week, randomised, double-blind trials. At endpoint, change from baseline on HAMD17 was greater in the duloxetine group (-7.0) than in the placebo group (-4.1) (p = 0.005). Response and remission rates, and improvement on the Clinical Global Impressions-Severity (CGI-S) scale, the Patient Global Impressions-Improvement (PGI-I) scale, and measures of painful symptoms were also significantly better in the duloxetine group (p < 0.05). Tolerability was consistent with that seen in previous studies of duloxetine in patients with more severe depression. In conclusion, duloxetine 60 mg/day is effective and well tolerated in milder MDD.

Unfolding of hen egg lysozyme by molecular dynamics simulations at 300K: insight into the role of the interdomain interface.

We present the results of two 1.2 ns molecular dynamics (MD) unfolding simulations on hen egg lysozyme in water at 300K, performed using a new procedure called PEDC (Path Exploration With Distance Constraints). This procedure allows exploration of low energy structures as a function of increasing RMSD from the native structure, and offers especially the possibility of extensive exploration of the conformational space during the initial unfolding stages. The two independent MD simulations gave similar chronology of unfolding events: disruption of the active site, kinking of helix C, partial unfolding of the three-stranded beta-sheet to a two-stranded sheet (during which the helices A, B, and D remain to a great extent native), and finally unfolding of the beta-domain and partial unfolding of the alpha-domain in which hydrophobic clusters persist. We show particularly that the loss of hydrophobic contacts between the beta-sheet turn residues Leu55 and Ile56 and the hydrobic patch of the alpha-domain destabilizes the beta-domain and leads to its unfolding, suggesting that the correct embedding of these residues in the alpha-beta interface may constitute the rate limiting step in folding. These results are in accord with experimental observations on the folding/unfolding behavior of hen egg lysozyme at room temperature. They would also explain the loss of stability and the tendency to aggregation observed for the mutant Leu55Thr, and the slow refolding kinetics observed in the analogous amyloidogenic variant of human lysozyme.

Tertiary and quaternary conformational changes in aspartate transcarbamylase: a normal mode study.

Aspartate transcarbamylase (ATCase) initiates the pyrimidine biosynthetic pathway in Escherichia coli. Binding of aspartate to this allosteric enzyme induces a cooperative transition between the tensed (T) and relaxed (R) states of the enzyme which involves large quaternary and tertiary rearrangements. The mechanisms of the transmission of the regulatory signal to the active site (60 A away) and that of the cooperative transition are not known in detail, although a large number of single, double, and triple site-specific mutants and chimeric forms of ATCase have been obtained and kinetically characterized. A previous analysis of the very low-frequency normal modes of both the T and R state structures of ATCase identified some of the large-amplitude motions mediating the intertrimer elongation and rotation that occur during the cooperative transition (Thomas et al., J. Mol. Biol. 257:1070-1087, 1996; Thomas et al., J. Mol. Biol. 261:490-506, 1996). As a complement to that study, the deformation of the quaternary and tertiary structure of ATCase by normal modes below 5 cm(-1) is investigated in this article. The ability of the modes to reproduce the domain motions occurring during the transition is analyzed, with special attention to the interdomain closure in the catalytic chain, which has been shown to be critical for homotropic cooperativity. The calculations show a coupling between the quaternary motions and more localized motions involving specific residues. The particular dynamic behavior of these residues is examined in the light of biochemical results to obtain insights into their role in the transmission of the allosteric signal.

1.2 A refinement of the Kunitz-type domain from the alpha3 chain of human type VI collagen.

The recombinant Kunitz-type domain (C5) of human collagen alpha3(VI) chain was previously described at 1.6 A resolution at room temperature. By changing the crystallization conditions and using synchrotron radiation, we are able to record diffraction data to 1.2 A resolution for crystals of the same space group at 291 K. The protein-water-ion model has been refined anisotropically against these new data using the program SHELXL93; the results converged to an R factor of 15.0%, with all data between 7 and 1.2 A. The final electron-density map reveals a clear chain tracing with a few disordered residues and five residues out of 58 that present alternate conformations. The Cys14-Cys38 bond presents the less frequently observed left-hand conformation (chi1 = -60 degrees). The solvent molecules and a phosphate ion are well ordered with an average B of 38 A2. The high-resolution structure reveals the N and C termini which were missing from the 1.6 A structure.

Conformational dynamics and enzyme activity.

Conformational flexibility and structural fluctuations play an important role in enzyme activity. A great variety of internal motions ranging over different time scales and of different amplitudes are involved in the catalytic cycle. These different types of motions and their functional consequences are considered in the light of experimental data and theoretical analyses. The conformational changes upon substrate binding, and particularly the hinge-bending motion which occurs in enzymes made of two domains, are analyzed from several well documented examples. The conformational events accompanying the different steps of the catalytic cycle are discussed. The last section concerns the motions involved in the allosteric transition which regulates the enzyme activity.

Analysis of the low-frequency normal modes of the R state of aspartate transcarbamylase and a comparison with the T state modes.

Aspartate transcarbamylase (ATCase) is a classic example of an allosteric enzyme. It catalyzes the conversion of aspartate to carbamyl aspartate, which is the first substrate in the biosynthesis of pyrimidines. Although ATCase is well characterized, both structurally and biochemically, little is known at the atomic level about the large amplitude motions that govern its T-->R quaternary transition. We present the results of calculations of the very-low-frequency normal modes of the CTP-ligated R state ATCase, and we compare them with the equivalent modes in the CTP-ligated T state ATCase. The large-amplitude, delocalized modes of frequencies below 4 cm-1 contribute a large fraction of the atomic fluctuations observed experimentally. They show some ability to drive the R-state structure towards the T-state structure, by promoting some of the quaternary structure rearrangements that take place during the allosteric process. Their potential role in the T-->R transition is quantified and compared with the role of the low-frequency modes of the T state in the quaternary rearrangement.

Motions in hemoglobin studied by normal mode analysis and energy minimization: evidence for the existence of tertiary T-like, quaternary R-like intermediate structures.

The normal mode analysis of human hemoglobin showed the presence in the deoxy T-state of one main preferential direction that brings the structure close to the R-state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate Tmin(d1), which is closer to R; the normal modes of Tmin(d1) indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to Tmin(d1), yielding Tmin(d2); then the procedure was repeated, yielding the intermediate structures Tmin(d3) and Tmin(d4). The structures Tmin(d2), Tmin(d3) and Tmin(d4) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, Tmin(d1), is different from the T and R structures, while the second set, Tmin(d2), Tmin(d3) and Tmin(d4), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha1 beta1 and alpha1 beta2 are R-like, and the alpha and beta heme environments are still T-like.

Analysis of the low frequency normal modes of the T-state of aspartate transcarbamylase.

Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli. It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally. As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist. In this work we have calculated the low frequency normal modes of the CTP-ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition. The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors. Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R-state structure is approached. The implication of these mode motions for the overall T-->R transition process is discussed.

Polar fluctuations in proteins: molecular-dynamic studies of cytochrome c in aqueous solution.

The equilibrium fluctuations (the polar fluctuations), of yeast cytochrome c are studied using nanosecond molecular-dynamic simulations in a spherical droplet of water, with a particular emphasis on the fluctuations of the total dipole moment, which determine the average relative permittivity. These fluctuations follow a simple probability distribution, predicted by continuum electrostatics, and already observed in simulations of several polar liquids. An important component consists of diffusive, mutually independent, motions of the charged side chains at the protein surface. A quasiharmonic normal mode analysis of the trajectory shows that while motions covering a large range of frequencies contribute to the polar fluctuations, the four lowest frequency modes account for 50% of the overall static relative permittivity of ca. 25. The fluctuations of the protein bulk, i.e. parts other than the charged side chains, are distributed over a larger number of modes. Modes up to at least 60 cm-1 contribute to the average relative permittivity of the protein interior of ca. 4. The water surrounding the protein, despite the structural perturbation represented by the protein, has fluctuations similar to pure water, consistent with the idea of a linear solvent response to the protein charges. The relationship between the microscopic fluctuations seen in the simulations and simple continuum models is discussed.

Computation of low-frequency normal modes in macromolecules: improvements to the method of diagonalization in a mixed basis and application to hemoglobin.

The method of diagonalization in a mixed basis (DIMB) that was published previously (Mouawad.), L. and Perahia D., Biopolymers 33, 599, 1993), allows the computation of the low-frequency vibrational modes for large macromolecules. Improvements to this method are presented here, namely the single and double truncation window techniques. The best convergence rate is obtained with the double truncation windows, which couple most efficiently the parts of the macromolecule which are far in sequence but close in space. Both methods were applied to the T-state of hemoglobin, to compare their efficiency. The resulting modes are analyzed in order to study the pathways between T- and R-states of this protein. They show that the quaternary conformational are mainly due to one mode at 2 cm-1.

Internal and interfacial dielectric properties of cytochrome c from molecular dynamics in aqueous solution.

The dielectric properties of proteins are central to their stability and activity. We use the Fröhlich-Kirkwood theory of dielectrics to analyze two 1-ns molecular dynamics simulations of ferro- and ferricytochrome c in spherical droplets of 1400 water molecules. Protein and solvent are idealized as a series of concentric, spherical, dielectric media. Analysis results depend strongly on the treatment of the charged protein side chains at the protein/solvent interface. If charged side chains are viewed as part of the protein medium, then the protein dipole fluctuations are dominated by large, mutually uncorrelated, anisotropic, motions of the charged side chains. It is then incorrect to view the protein region as a single, homogeneous dielectric material. If one does take this view, estimates of the protein "dielectric constant" vary from 16 to 37, depending on the exact choice of model parameters. In contrast, if the charged portions of the charged side chains are viewed as part of the solvent medium, then theory and simulation are consistent: the protein dipole fluctuations excluding charged side chains are roughly those of a homogeneous, isotropic dielectric medium, with a dielectric constant of 4.7 +/- 1.0 (ferro) or 3.4 +/- 1.0 (ferri), in agreement with powder experiments. Statistical uncertainty and sensitivity to model parameters are small. Analysis of the radial dependence of the dipole fluctuations suggests that the inner half of the protein has a somewhat lower dielectric constant of 1.5-2, consistent with its biological function in electron transfer. These results suggest that Poisson-Boltzmann models could treat the protein bulk as a low-dielectric medium and the charged surface groups as part of the solvent region.

A free-energy simulation study of a bacterial collagenase inhibitor.

The cis-trans conformational isomerisation of the N-methylated peptide bond of a bacterial collagenases peptide inhibitor (HS-CH2-CH2-CO-Pro-NMe-Ala) has been investigated by molecular dynamics simulations, energy minimisations and free-energy simulations in presence of solvent molecules. The free-energy difference between the cis and trans forms obtained by the thermodynamics integration method is equal to 0.95 kcal/mol in favour of the trans form, in accord with the experimental result. The main contribution to this free-energy difference comes from solute-water electrostatic interactions. Interestingly, we show that the number of interactions between water molecules and the oxygen atoms of the inhibitor is larger in the trans form than in the cis. Thus the organisation of water molecules around the inhibitor appears crucial in determining the population of the cis and trans conformers.