Molecular dynamics simulation of shear- and stretch-induced dissociation of P-selectin/PSGL-1 complex

By mediating the tethering and rolling of leukocytes on vascular surfaces, the interactions between P-selectin and the P-selectin glycoprotein ligand 1 (PSGL-1) play crucial roles during inflammation cascade. Tensile stretch produced by rolling leukocytes and shear stress exerted by blood flow constitute the two types of mechanical forces that act on the P-selectin/PSGL-1 bond. These forces modulate not only dissociation kinetics of this bond, but also the leukocyte adhesion dynamics. However, the respective contribution of the two forces to bond dissociation and to the corresponding microstructural bases remains unclear. To mimic the mechanical microenvironment, we developed two molecular dynamics approaches; namely, an approach involving the shear flow field with a controlled velocity gradient, and the track dragging approach with a defined trajectory. With each approach or with both combined, we investigate the microstructural evolution and dissociation kinetics of the P-LE/SGP-3 construct, which is the smallest functional unit of the P-selectin/PSGL-1 complex. The results demonstrate that both shear flow and tensile stretch play important roles in the collapse of the construct and that, before bond dissociation, the former causes more destruction of domains within the construct than the latter. Dissociation of the P-LE/SGP-3 construct features intramolecular destruction of the epidermal-growth-factor (EGF) domain and the breaking of hydrogen-bond clusters at the P-selectin-lectin/EGF interface. Thus, to better understand how mechanics impacts the dissociation kinetics of the P-selectin/PSGL-1 complex, we propose herein two approaches to mimic its physiological mechanical environment.     

A structure-based sliding-rebinding mechanism for catch bonds

Catch bonds, whose lifetimes are prolonged by force, have been observed in selectin-ligand interactions and other systems. Several biophysical models have been proposed to explain this counterintuitive phenomenon, but none was based on the structure of the interacting molecules and the noncovalent interactions at the binding interface. Here we used molecular dynamics simulations to study changes in structure and atomic-level interactions during forced unbinding of P-selectin from P-selectin glycoprotein ligand-1. A mechanistic model for catch bonds was developed based on these observations. In the model, “catch” results from forced opening of an interdomain hinge that tilts the binding interface to allow two sides of the contact to slide against each other. Sliding promotes formation of new interactions and even rebinding to the original state, thereby slowing dissociation and prolonging bond lifetimes. Properties of this sliding-rebinding mechanism were explored using a pseudoatom representation and Monte Carlo simulations. The model has been supported by its ability to fit experimental data and can be related to previously proposed two-pathway models.     

Molecular dynamics simulations of Factor Xa: insight into conformational transition of its binding subsites

Protein flexibility and conformational diversity is well known to be a key characteristic of the function of many proteins. Human blood coagulation proteins have multiple substrates, and various protein-protein interactions are required for the smooth functioning of the coagulation cascade to maintain blood hemostasis. To address how a protein may cope with multiple interactions with its structurally diverse substrates and the accompanied structural changes that may drive these changes, we studied human Factor X. We employed 20 ns of molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on two different conformational forms of Factor X, open and closed, and observed an interchangeable conformational transition from one to another. This work also demonstrates the roles of various aromatic residues involved in aromatic-aromatic interactions, which make this dynamic transition possible.     

Simulating force-induced conformational transitions in polysaccharides with the SMD replica exchange method

Conventional steered molecular dynamics (SMD) simulations do not readily reproduce equilibrium conditions of atomic force microscopy (AFM) stretch and release measurements of polysaccharides undergoing force-induced conformational transitions because of the gap between the timescales of computer simulations ( approximately 1 mus) and AFM measurements ( approximately 1 s). To circumvent this limitation, we propose using the replica exchange method (REM) to enhance sampling during SMD simulations. By applying REM SMD to a small polysaccharide system and comparing the results with those from AFM stretching experiments, we demonstrate that REM SMD reproduces the experimental results not only qualitatively but quantitatively, approaching near equilibrium conditions of AFM measurements. As tested in this work, hysteresis and computational time of REM SMD simulations of short polysaccharide chains are significantly reduced as compared to regular SMD simulations, making REM SMD an attractive tool for studying forced-induced conformational transitions of small biopolymer systems.     

Force-based analysis of multidimensional energy landscapes: application of dynamic force spectroscopy and steered molecular dynamics simulations to an antibody fragment-peptide complex

Multidimensional energy landscapes are an intrinsic property of proteins and define their dynamic behavior as well as their response to external stimuli. In order to explore the energy landscape and its implications on the dynamic function of proteins dynamic force spectroscopy and steered molecular dynamics (SMD) simulations have proved to be important tools. In this study, these techniques have been employed to analyze the influence of the direction of the probing forces on the complex of an antibody fragment with its peptide antigen. Using an atomic force microscope, experiments were performed where the attachment points of the 12 amino acid long peptide antigen were varied. These measurements yielded clearly distinguishable basal dissociation rates and potential widths, proving that the direction of the applied force determines the unbinding pathway. Complementary atomistic SMD simulations were performed, which also show that the unbinding pathways of the system are dependent on the pulling direction. However, the main barrier to be crossed was independent of the pulling direction and is represented by a backbone hydrogen bond between Gly^H-H40 of the antibody fragment and Glu^Oε-6_peptide of the peptide. For each pulling direction, the observed barriers can be correlated with the rupture of specific interactions, which stabilize the bound complex. Furthermore, although the SMD simulations were performed at loading rates exceeding the experimental rates by orders of magnitude due to computational limitations, a detailed comparison of the barriers that were overcome in the SMD simulations with the data obtained from the atomic force microscope unbinding experiments show excellent agreement.     

Forced Unfolding of Apocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> by Steered Molecular Dynamics Simulation

Apocytochrome b5 (apocyt b5), a small b-type cytochrome with heme prosthetic group removal, has been subjected to steered molecular dynamics (SMD) simulations for investigating the consequences of mechanical force-induced unfolding. Both constant velocity (0.5 and 1.0 A/ps) and constant force (500, 750 and 1000 pN) stretching have been employed to model forced unfolding of apocyt b5. The results of SMD simulations elucidate that apocyt b5 is protected against external stress mainly through the interstrand hydrogen bonding between its beta1-beta2 and beta2-beta3 strands, highlighting the importance of hydrophobic core 2 in stabilization of apocyt b5. The existence of intermediate states manifested by current simulations in the forced unfolding pathway of apocyt b5 is different from the observations in pervious thermal or chemical unfolding studies in the absence of force. The present study could thus provide insights into the relationship between the two cooperative functional modules of apocyt b5 and also guide the rational molecular design of heme proteins.     

Molecular Basis for the Dissociation Dynamics of Protein A-Immunoglobulin G1 Complex

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.     

Steered molecular dynamics simulation of the binding of the β2 and β3 regions in domain-swapped human cystatin C dimer

The crystal structure of the human cystatin C (hCC) dimer revealed that a stable twofold-symmetric dimer was formed via 3D domain swapping. Domain swapping with the need for near-complete unfolding has been proposed as a possible route for amyloid fibril initiation. Thus, the interesting interactions that occur between the two molecules may be important for the further aggregation of the protein. In this work, we performed steered molecular dynamics (SMD) simulations to investigate the dissociation of the β2 and β3 strands in the hCC dimer. The energy changes observed during the SMD simulations showed that electrostatic interactions were the dominant interactions involved in stabilizing the two parts of the dimer during the early stages of SMD simulation, whereas van der Waals (VDW) interactions and electrostatic interactions were equally matched during the latter stages. Furthermore, our data indicated that the two parts of the dimer are stabilized by intermolecular hydrogen bonds among the residues Arg51 (β2), Gln48 (β2), Asp65 (β3), and Glu67 (β3), salt bridges among the residues Arg53 (β2), Arg51 (β2), and Asp65 (β3), and VDW interactions among the residues Gln48 (β2), Arg51 (β2), Glu67 (β3), Asp65 (β3), Phe63 (β3), and Asn61 (β3). The residues Gln48 (β2), Arg51 (β2), Asp65 (β3) and Glu67 (β3) appear to be crucial, as they play important roles in both electrostatic and VDW interactions. Thus, the present study determined the key residues involved in the stabilization of the domain-swapped dimer structure, and also provided molecular-level insights into the dissociation process of the hCC dimer.     

Evaluation of intermolecular forces in a circulating system

Intercellular interactions, which are mediated by a variety of complex intercellular molecules through the processes of formation and dissociation of molecular bonds, play a critical role in regulating cellular functions in biological systems. Various approaches are applied to evaluate intercellular or molecular bonding forces. To quantify the intermolecular interaction forces, flow chamber has become a meaningful technique as it can ultimately mimic the cellular microenvironment in vivo under physiological flow conditions. Hydrodynamic forces are usually used to predict the intercellular forces down to the single molecular level. However, results show that only using hydrodynamic force will overestimate up to 30% of the receptor-ligand strength when the non-specific forces such as Derjaguin-Landau-Verway-Overbeek (DLVO) forces become un-neglected. Due to the nature of high ion concentration in the physiological condition, electrostatic force is largely screened which will cause DLVO force unbalanced. In this study, we propose to take account of the DLVO force, including van der Waals (VDW) force and electrostatic force, to predict the intermolecular forces of a cell doublet and cell-substrate model in a circulating system. Results also show that the DLVO force has a nonlinear effect as the cell-cell or cell-substrate distance changes. In addition, we used the framework of high accuracy hydrodynamic theories proved in colloidal systems. It is concluded that DLVO force could not be ignored in quantitative studies of molecular interaction forces in circulating system. More accurate prediction of intercellular forces needs to take account of both hydrodynamic force and DLVO force.     

Effect of protonation of the N-acetyl neuraminic acid residue of sialyl Lewis(X): a molecular orbital study with insights into its binding properties toward the carbohydrate recognition domain of E-selectin

Semiempirical molecular orbital (MO) calculations with an implicit treatment of the water environment were employed in order to assess whether the sialyl Lewis(X) (sLe(X)) tetrasaccharide binds to E-selectin in the anionic or neutral (i.e., protonated) state. The analysis of the frontier molecular orbitals, electrostatic potential surfaces, and conformational behavior of the sugar indicates that its neutral form possesses the necessary characteristics for binding. In particular, the LUMO level of the neutral sLe(X) molecule is localized both on the carboxylic group of the N-acetyl neuraminic acid (NeuNAc) residue and on the nearby glycosidic linkage. These two moieties interact with the Arg97 residue of E-selectin, as revealed by a recent crystal structure analysis of the E-selectin/sLe(X) complex. The energetics of this specific interaction was investigated with the aid of ab initio Hartree-Fock MO calculations, which resulted in a BSSE-corrected binding energy of 16.63 kcal/mol. Our observations could open up new perspectives in the design of sLe(X) mimics.     

Mechanical biochemistry of proteins one molecule at a time

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells (and by extension, that of the multicellular organisms they form) is the result of cycles of mechanosensing, mechanotransduction, and mechanoresponse. Recently developed single-molecule atomic force microscopy techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins. Single-molecule mechanical techniques are providing fundamental information on the structure and function of proteins and are becoming an indispensable tool to understand how these molecules fold and work.     

Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force

Integrin alpha(V)beta(3) binds to extracellular matrix proteins through the tripeptide Arg-Gly-Asp (RGD), forming a shallow crevice rather than a deep binding pocket. A dynamic picture of how the RGD-alpha(V)beta(3) complex resists dissociation by mechanical force is derived here from steered molecular dynamic (SMD) simulations in which the major force peak correlates with the breaking of the contact between Asp(RGD) and the MIDAS ion. SMD predicts that the RGD-alpha(V)beta(3) complex is stabilized from dissociation by a single water molecule tightly coordinated to the divalent MIDAS ion, thereby blocking access of free water molecules to the most critical force-bearing interaction. The MIDAS motif is common to many other proteins that contain the phylogenetically ancient von Willebrand A (vWA) domain. The functional role of single water molecules tightly coordinated to the MIDAS ion might reflect a general strategy for the stabilization of protein-protein adhesion against cell-derived forces through divalent cations.     

Biomechanics of leukocyte rolling

Leukocyte rolling on endothelial cells and other P-selectin substrates is mediated by P-selectin binding to P-selectin glycoprotein ligand-1 expressed on the tips of leukocyte microvilli. Leukocyte rolling is a result of rapid, yet balanced formation and dissociation of selectin-ligand bonds in the presence of hydrodynamic shear forces. The hydrodynamic forces acting on the bonds may either increase (catch bonds) or decrease (slip bonds) their lifetimes. The force-dependent 'catch-slip' bond kinetics are explained using the 'two pathway model' for bond dissociation. Both the 'sliding-rebinding' and the 'allosteric' mechanisms attribute 'catch-slip' bond behavior to the force-induced conformational changes in the lectin-EGF domain hinge of selectins. Below a threshold shear stress, selectins cannot mediate rolling. This 'shear-threshold' phenomenon is a consequence of shear-enhanced tethering and catch bond-enhanced rolling. Quantitative dynamic footprinting microscopy has revealed that leukocytes rolling at venular shear stresses (>0.6 Pa) undergo cellular deformation (large footprint) and form long tethers. The hydrodynamic shear force and torque acting on the rolling cell are thought to be synergistically balanced by the forces acting on tethers and stressed microvilli, however, their relative contribution remains to be determined. Thus, improvement beyond the current understanding requires in silico models that can predict both cellular and microvillus deformation and experiments that allow measurement of forces acting on individual microvilli and tethers.     

Contribution of the CR Domain to P-Selectin Lectin Domain Allostery by Regulating the Orientation of the EGF Domain

The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS) combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1’ state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain.     

Dynamics fingerprint and inherent asymmetric flexibility of a cold-adapted homodimeric enzyme. A case study of the Vibrio alkaline phosphatase

Background

Protein dynamics influence protein function and stability and modulate conformational changes. Such motions depend on the underlying networks of intramolecular interactions and communicating residues within the protein structure. Here, we provide the first characterization of the dynamic fingerprint of the dimeric alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 (VAP), which is among the APs with the highest known k_cat at low temperatures.

Methods

Multiple all-atom explicit solvent molecular dynamics simulations were employed in conjunction with different metrics to analyze the dynamical patterns and the paths of intra- and intermolecular communication.

Results

Interactions and coupled motions at the interface between the two VAP subunits have been characterized, along with the networks of intramolecular interactions. It turns out a low number of intermolecular interactions and coupled motions, which result differently distributed in the two monomers. The paths of long-range communication mediated from the catalytic residues to distal sites were also characterized, pointing out a different information flow in the two subunits.

Conclusions

A pattern of asymmetric flexibility is evident in the two identical subunits of the VAP dimer that is intimately linked to a different distribution of intra- and intermolecular interactions. The asymmetry was also evident in pairs of cross-correlated residues during the dynamics.

General significance

The results here discussed provide a structural rationale to the half-of-site mechanism previously proposed for VAP and other APs, as well as a general framework to characterize asymmetric dynamics in homomeric enzymes.     

Molecular basis of lateral force spectroscopy nano-diagnostics: computational unbinding of autism related chemokine MCP-1 from IgG antibody

Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, is a potent chemoattractant of T cells and monocytes, involved in inflammatory and angio-proliferative brain and retinal diseases. Higher expression of MCP-1 is observed in metastatic tumors. Unusual levels of MCP-1 in the brain may be correlated with autism. Immunochemistry where atomic force microscope (AFM) tips functionalized with appropriate antibodies against MCP-1 are used could in principle support medical diagnostics. Useful signals from single molecule experiments may be generated if interaction forces are large enough. The chemokine-antibody unbinding force depends on a relative motion of the interacting fragments of the complex. In this paper the stability of the medically important MCP-1- immunoglobulin G antibody Fab fragment complex has been studied using steered molecular dynamics (SMD) computer simulations with the aim to model possible arrangements of nano-diagnostics experiments. Using SMD we confirm that molecular recognition in MCP1-IgG is based mainly on six pairs of residues: Glu39A - Arg98H, Lys56A - Asp52H, Asp65A - Arg32L, Asp68A - Arg32L, Thr32A - Glu55L, Gln61A - Tyr33H. The minimum external force required for mechanical dissociation of the complex depends on a direction of the force. The pulling of the MCP-1 antigen in the directions parallel to the antigen-antibody contact plane requires forces about 20 %–40 % lower than in the perpendicular one. Fortunately, these values are large enough that the fast lateral force spectroscopy may be used for effective nano-diagnostics purposes. We show that molecular modeling is a useful tool in planning AFM force spectroscopy experiments.

Dielectric screening effect of electronic polarization and intramolecular hydrogen bonding

Recent site‐resolved hydrogen exchange measurements have uncovered significant discrepancies between simulations and experimental data during protein folding, including the excessive intramolecular hydrogen bonds in simulations. This finding indicates a possibility that intramolecular charge–charge interactions have not included sufficient dielectric screening effect of the electronic polarization. Scaling down peptide atomic charges according to the optical dielectric constant is tested in this study. As a result, the number of intramolecular hydrogen bonds is lower than using unscaled atomic charges while reaching the same levels of helical contents or β‐hairpin backbone hydrogen bonds, because van der Waals interactions contribute substantially to peptide folding in water. Reducing intramolecular charge–charge interactions and hydrogen bonding increases conformational search efficiency. In particular, it reduces the equilibrium helical content in simulations using AMBER force field and the energy barrier in folding simulations using CHARMM force field.     

Steered molecular dynamics simulations on the binding of the appendant structure and helix-β2 in domain-swapped human cystatin C dimer

We have performed steered molecular dynamics (SMD) simulations to investigate the dissociation process between the appendant structure (AS) and helix-β2 in human cystatin C dimer. Energy change during SMD showed that electrostatic interactions, including hydrogen bonds and salt bridges, were the dominant interactions to stabilize the two parts of the dimer. Furthermore, our data indicated that residues, Asn35, Asp40, Ser44, Lys75, and Arg93 play significant roles in the formation of these electrostatic interactions. Docking studies suggested that the interactions between AS and β2-helix were formed following domain swapping and were responsible for stabilizing the structure of the domain-swapped dimer.     

Steered molecular dynamics simulations on the binding of the appendant structure and helix-β2 in domain-swapped human cystatin C dimer

We have performed steered molecular dynamics (SMD) simulations to investigate the dissociation process between the appendant structure (AS) and helix-β2 in human cystatin C dimer. Energy change during SMD showed that electrostatic interactions, including hydrogen bonds and salt bridges, were the dominant interactions to stabilize the two parts of the dimer. Furthermore, our data indicated that residues, Asn35, Asp40, Ser44, Lys75, and Arg93 play significant roles in the formation of these electrostatic interactions. Docking studies suggested that the interactions between AS and β2-helix were formed following domain swapping and were responsible for stabilizing the structure of the domain-swapped dimer.     

Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation

Protein engineering Phi-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI I27. TNfn3 and TI I27 are members of different protein superfamilies and have no sequence identity but they have the same beta-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI I27. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI I27, TNfn3 unfolds via a force-stabilised intermediate. The key event in forced unfolding in TI I27 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical Phi-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI I27, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force.