A molecular dynamics study and free energy analysis of complexes between the Mlc1p protein and two IQ motif peptides

The Mlc1p protein from the budding yeast Saccharomyces cerevisiae is a Calmodulin-like protein, which interacts with IQ-motif peptides located at the yeast's myosin neck. In this study, we report a molecular dynamics study of the Mlc1p-IQ2 protein-peptide complex, starting with its crystal structure, and investigate its dynamics in an aqueous solution. The results are compared with those obtained by a previous study, where we followed the solution structure of the Mlc1p-IQ4 protein-peptide complex by molecular dynamics simulations. After the simulations, we performed an interaction free-energy analysis using the molecular mechanics Poisson-Boltzmann surface area approach. Based on the dynamics of the Mlc1p-IQ protein-peptide complexes, the structure of the light-chain-binding domain of myosin V from the yeast S. cerevisiae is discussed.     

Myosin V movement: lessons from molecular dynamics studies of IQ peptides in the lever arm

Myosin V moves along actin filaments by an arm-over-arm motion, known as the lever mechanism. Each of its arms is composed of six consecutive IQ peptides that bind light chain proteins, such as calmodulin or calmodulin-like proteins. We have employed a multistage approach in order to investigate the mechanochemical structural basis of the movement of myosin V from the budding yeast Saccharomyces cerevisiae. For that purpose, we previously carried out molecular dynamics simulations of the Mlc1p-IQ2 and the Mlc1p-IQ4 protein-peptide complexes, and the present study deals with the structures of the IQ peptides when stripped from the Mlc1p protein. We have found that the crystalline structure of the IQ2 peptide retains a stable rodlike configuration in solution, whereas that of the IQ4 peptide grossly deviates from its X-ray conformation exhibiting an intrinsic tendency to curve and bend. The refolding process of the IQ4 peptide is initially driven by electrostatic interactions followed by nonpolar stabilization. Its bending appears to be affected by the ionic strength, when ionic strength higher than approximately 300 mM suppresses it from flexing. Considering that a poly-IQ sequence is the lever arm of myosin V, we suggest that the arm may harbor a joint, localized within the IQ4 sequence, enabling the elasticity of the neck of myosin V. Given that a poly-IQ sequence is present at the entire class of myosin V and the possibility that the yeast's myosin V molecule can exist either as a nonprocessive monomer or as a processive dimer depending on conditions (Krementsova, E. B., Hodges, A. R., Lu, H., and Trybus, K. M. (2006) J. Biol. Chem. 281, 6079-6086), our observations may account for a general structural feature for the myosins' arm embedded flexibility.     

Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs-functional implications

IQ motifs are widespread in nature. Mlc1p is a calmodulin-like myosin light chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP-related protein, Iqg1p, playing a role in polarized growth and cytokinesis in Saccharomyces cerevisiae. The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically. When bound to IQ2, Mlc1p adopts a compact conformation in which both the N- and C-lobes interact with the IQ motif. However, in the complex with IQ4, the N-lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p. The two light chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N-lobes of calmodulin-like light chains. The correlation between light chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p. The resulting 'free' N-lobes of myosin light chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment.     

Molecular dynamics study of the human insulin B peptide SHLVEALYLVCGERGG complexed with HLA-DQ8 reveals important hydrogen bond interactions

The basis of proper recognition of pathogens and tumours is provided by adaptive immunity. This immunological reaction of the recognition function of T-cell receptors on T lymphocytes detects antigenic peptides bound to major histocompatibility complex (MHC) molecules. Structural insight into this process has few grown considerably in the last years. In some of the cases, antigens are self-protein fragments causing autoimmunity diseases. Type 1 diabetes is such a disease connected with the human leukocyte antigen-DQ8 molecule, a class II MHC glycoprotein. Its crystal structure, complexed with LVEALYLVCGERGG peptide (insulin B peptide), has been solved, and important information about the significance of P1, P4 and P9 binding pockets has been discovered. The complex structure also revealed an unusual large number of intermolecular hydrogen bonds between insulin B peptide and MHC molecule. To further investigate the dynamics of peptide/MHC interactions, we perform molecular dynamic simulations in explicit water. Analysis of the results provided useful information of the binding of the peptide antigen to MHC molecule, which is supported by numerous hydrogen bonds besides the electrostatic (P1 and P9 pockets) or hydrophobic interactions (P4). Results also allowed some implications to be drawn for the role of residues located outside of the binding groove.     

Theoretical studies of the structure and molecular dynamics of a peptide crystal

Energy minimizations and molecular dynamics simulations have been performed on the cyclic peptide cyclo-(Ala-Pro-D-Phe)2 in both the isolated and crystal states. The results of these calculations have been analyzed, both to investigate our ability to reproduce experimental data (structure and vibrational and NMR spectra) and to investigate the effects of environment on the energy, structure, and dynamics of peptides. Comparison of the minimized and time-averaged crystal systems with the experimental peptide structure shows that the calculations have closely reproduced the experimental structure. Molecular dynamics of the isolated molecule has led to a new conformation, which is approximately equal to 8.5 kcal/mol more stable than the conformation that exists in the crystal, the latter conformation being stabilized by intermolecular (packing) forces. This illustrates the considerable effect that environment can have on the conformation of peptides. The crystal environment has also been shown to significantly reduce the dynamic conformational fluctuations seen for the isolated molecule. The behavior of the peptide during the isolated simulation also supports the experimental NMR observation of a symmetric structure that differs from the asymmetric, instantaneous structures which characterize the molecule during the dynamics. Calculations of vibrational frequencies of the peptide in the crystal and isolated states show the expected shifts in bond-stretching frequencies due to intermolecular interactions. Finally, we have calculated NMR coupling constants from the dynamics simulation of the isolated peptide and have compared these with the experimental values. This has led to a possible reinterpretation of the experimental data.     

Structural basis for the interaction of the myosin light chain Mlc1p with the myosin V Myo2p IQ motifs

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.     

Enhanced sampling protocol to elucidate fusion peptide opening of SARS-CoV-2 spike protein

Large-scale conformational transitions in the spike protein S2 domain are required during host-cell infection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Although conventional molecular dynamics simulations have been extensively used to study therapeutic targets of SARS-CoV-2, it is still challenging to gain molecular insight into the key conformational changes because of the size of the spike protein and the long timescale required to capture these transitions. In this work, we have developed an efficient simulation protocol that leverages many short simulations, a dynamic selection algorithm, and Markov state models to interrogate the structural changes of the S2 domain. We discovered that the conformational flexibility of the dynamic region upstream of the fusion peptide in S2 is coupled to the proteolytic cleavage state of the spike protein. These results suggest that opening of the fusion peptide likely occurs on a submicrosecond timescale after cleavage at the S2′ site. Building on the structural and dynamical information gained to date about S2 domain dynamics, we provide proof of principle that a small molecule bound to a seam neighboring the fusion peptide can slow the opening of the fusion peptide, leading to a new inhibition strategy for experiments to confirm. In aggregate, these results will aid the development of drug cocktails to inhibit infections caused by SARS-CoV-2 and other coronaviruses.     

Molecular dynamics simulations of a pulmonary surfactant protein B peptide in a lipid monolayer

Pulmonary surfactant is a complex mixture of lipids and proteins that lines the air/liquid interface of the alveolar hypophase and confers mechanical stability to the alveoli during the breathing process. The desire to formulate synthetic mixtures for low-cost prophylactic and therapeutic applications has motivated the study of the specific roles and interactions of the different components. All-atom molecular dynamics simulations were carried out on a model system composed of a monolayer of palmitic acid (PA) and a surfactant protein B peptide, SP-B(1-25). A detailed structural characterization as a function of the lipid monolayer specific area revealed that the peptide remains inserted in the monolayer up to values of specific area corresponding to an untilted condensed phase of the the pure palmitic acid monolayer. The system remains stable by altering the conformational order of both the anionic lipid monolayer and the peptide secondary structure. Two elements appear to be key for the constitution of this phase: an electrostatic interaction between the cationic peptide residues with the anionic headgroups, and an exclusion of the aromatic residues on the hydrophobic end of the peptide from the hydrophilic and aqueous regions.     

Conformational study of cyclolinopeptide A. A distance geometry and molecular dynamics approach

The conformation of cyclolinopeptide A, c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of distance geometry calculations and molecular dynamics simulations. The starting points for all the calculations were an X-ray structure and other structures obtained from distance geometry calculations based on NMR data. Restrained and unrestrained molecular dynamics simulations are reported in vacuo and in CCl4. Structural and dynamic properties are investigated and compared with those experimentally determined. The conformation obtained from the MD simulations which best reproduces the NMR parameters is at the same time one of the most stable ones and is also fairly similar to the crystal structure. An explanation for the occurrence of multiple conformations in solution at room temperature is given.     

Conformational flexibility of the MHC class I alpha1-alpha2 domain in peptide bound and free states: a molecular dynamics simulation study

Major histocompatibility complex class I proteins play a key role in the recognition and presentation of peptide antigens to the host immune system. The structure of various major histocompatibility complex class I proteins has been determined experimentally in complex with several antigenic peptides. However, the structure in the unbound (empty) form is not known. To study the conformational dynamics of the empty major histocompatibility complex class I molecule comparative molecular dynamics simulations have been performed starting from the crystal structure of a peptide bound class I peptide-binding domain in the presence and absence of a peptide ligand. Simulations including the bound peptide stayed close to the experimental start structure at both simulation temperatures (300 and 355 K) during the entire simulation of 26 ns. Several independent simulations in the absence of peptide indicate that the empty domain may not adopt a single defined conformation but is conformationally significantly more heterogeneous in particular within the alpha-helices that flank the peptide binding cleft. The calculated conformational dynamics along the protein chain correlate well with available spectroscopic data and with the observed site-specific sensitivity of the empty class I protein to proteolytic digestion. During the simulations at 300 K the binding region for the peptide N-terminus stayed close to the conformation in the bound state, whereas the anchor region for the C-terminus showed significantly larger conformational fluctuations. This included a segment at the beginning of the second alpha-helix in the domain that is likely to be involved in the interaction with the chaperone protein tapasin during the peptide-loading process. The simulation studies further indicate that peptide binding at the C- and N-terminus may follow different mechanisms that involve different degrees of induced conformational changes in the peptide-binding domain. In particular binding of the peptide C-terminus may require conformational stabilization by chaperone proteins during peptide loading.     

GTP drives myosin light chain 1 interaction with the class V myosin Myo2 IQ motifs via a Sec2 RabGEF-mediated pathway

The yeast myosin light chain 1 (Mlc1p) belongs to a branch of the calmodulin superfamily and is essential for vesicle delivery at the mother-bud neck during cytokinesis due to is ability to bind to the IQ motifs of the class V myosin Myo2p. While calcium binding to calmodulin promotes binding/release from the MyoV IQ motifs, Mlc1p is unable to bind calcium and the mechanism of its interaction with target motifs has not been clarified. The presence of Mlc1p in a complex with the Rab/Ypt Sec4p and with Myo2p suggests a role for Mlc1p in regulating Myo2p cargo binding/release by responding to the activation of Rab/Ypt proteins. Here we show that GTP or GTPgammaS potently stimulate Mlc1p interaction with Myo2p IQ motifs. The C-terminus of the Rab/Ypt GEF Sec2p, but not Sec4p activation, is essential for this interaction. Interestingly, overexpression of constitutively activated Ypt32p, a Rab/Ypt protein that acts upstream of Sec4p, stimulates Mlc1p/Myo2p interaction similarly to GTP although a block of Ypt32 GTP binding does not completely abolish the GTP-mediated Mlc1p/Myo2p interaction. We propose that Mlc1p/Myo2p interaction is stimulated by a signal that requires Sec2p and activation of Ypt32p.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.     

Reducing CDK4/6-p16(INK4a) interface. Computational alanine scanning of a peptide bound to CDK6 protein

The tumor suppressor gene p16INK4a is commonly found altered in numerous and different types of cancer. The encoded protein arrests cell cycle in G1 phase by binding to CDK4 and CDK6, inhibiting their kinase function. In 1995, a 20-residue peptide, extracted from p16INK4a protein sequence, was discovered that retains the cell cycle inhibition properties of the endogenous tumor suppressor. However, its structure has not been determined yet. In this article, the features of a theoretical structure of the peptide bound to CDK6 are reported. The complex was modeled from CDK6-p16INK4a X-ray crystal structure and through molecular dynamics. Final structure was assessed by comparing computed binding free energy changes, when single-alanine substitutions were brought about on the peptide, to experimental data. Better concordance was obtained when including a high level of solvation effects. Solute-solvent vdW energy and electrostatic energy between solute and first shells of water, computed through a force field and considering explicit waters, were also to be included to achieve reasonably good concordance between theoretical and experimental data.     

Change in conformation by DNA-peptide association: molecular dynamics of the Hin-recombinase-hixL complex.

The Hin-DNA complex is a molecular complex formed by the C-terminal 52mer peptide of the Hin-recombinase and a synthetic 13-bp hixL DNA. The peptide has three alpha-helices, the second and third of which form the helix-turn-helix motif to bind to the major groove. Both termini of the peptide reside within the minor groove. Three molecular dynamics simulations were performed based on the crystal structure of the Hin-DNA complex: one for the free Hin peptide, one for the free hixL DNA, and one for the complex. Analyses of the trajectories revealed that the dynamic fluctuations of both the Hin peptide and the hixL DNA were lowered by the complex formation. The simulation supported the experimental observation that the N-terminus and the helix-turn-helix motif were critical for formation of the complex, but the C-terminus played only a supportive role in DNA recognition. The simulations strongly suggested that the binding reaction should proceed by the induced fit mechanism. The ion and solvent distributions around the molecules were also examined.     

A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation

Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring [1] [2] [3], which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles. Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring [4] [5] [6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck [6]. We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins. Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p. These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p.     

Enhanced sampling of peptide and protein conformations using replica exchange simulations with a peptide backbone biasing-potential

During replica exchange molecular dynamics (RexMD) simulations, several replicas of a system are simulated at different temperatures in parallel allowing for exchange between replicas at frequent intervals. This technique allows significantly improved sampling of conformational space and is increasingly being used for structure prediction of peptides and proteins. A drawback of the standard temperature RexMD is the rapid increase of the replica number with increasing system size to cover a desired temperature range. In an effort to limit the number of replicas, a new Hamiltonian-RexMD method has been developed that is specifically designed to enhance the sampling of peptide and protein conformations by applying various levels of a backbone biasing potential for each replica run. The biasing potential lowers the barrier for backbone dihedral transitions and promotes enhanced peptide backbone transitions along the replica coordinate. The application on several peptide cases including in all cases explicit solvent indicates significantly improved conformational sampling when compared with standard MD simulations. This was achieved with a very modest number of 5-7 replicas for each simulation system making it ideally suited for peptide and protein folding simulations as well as refinement of protein model structures in the presence of explicit solvent.     

Cooperative folding mechanism of a beta-hairpin peptide studied by a multicanonical replica-exchange molecular dynamics simulation

G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific beta-hairpin within 6 micros. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a multicanonical replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like beta-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high phi-values of the turn region observed by experiment.     

Exploring CRD mobility during RAS/RAF engagement at the membrane

During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3–5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.