Paratope and epitope mapping of the antithrombotic antibody 6B4 in complex with platelet glycoprotein Ibalpha

The monoclonal antibody 6B4 has a potent antithrombotic effect in nonhuman primates by binding to the flexible loop, also known as the beta-switch region (amino acids 230-242), of glycoprotein Ibalpha (GPIbalpha). This interaction blocks, in high shear stress conditions, the specific interaction between GPIbalpha and von Willebrand factor suppressing platelet deposition to the damaged vessel wall, a key event in the pathogenesis of arterial thrombosis. To understand the interactions between this antibody and its antigen at the amino acid level, we here report the identification of the paratope and epitope in 6B4 and GPIbalpha, respectively, by using computer modeling and site-directed mutagenesis. The docking programs ZDOCK (rigid body docking) and HADDOCK (flexible docking) were used to model the interaction of 6B4 with GPIbalpha and to delineate the respective paratope and epitope. 6B4 and GPIbalpha mutants were constructed and assayed for their capacity to bind GPIbalpha and 6B4, respectively. From these data, it is found that the paratope of 6B4 is mainly formed by five residues: Tyr(27D), Lys(27E), Asp(28), and Glu(93) located in light chain CDR1 and -3, respectively, and Tyr(100C) of the heavy chain CDR3. These residues form a valley, where the GPIbalpha flexible loop can bind via residues Asp(235) and Lys(237). The experimental results were finally used to build a more accurate docking model. Taken together, this information provides guidelines for the design of new derivatized lead compounds with antithrombotic properties.     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Energetic analysis of an antigen/antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction.

Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of deltadeltaG(HyHEL-10_mutant) vs. deltadeltaG(HEWL_mutant) for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32(H)N/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the deltadeltaG values of the Tyr --> Phe vs. the corresponding Tyr --> Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total deltadeltaG observed for the Tyr --> Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31D(L)/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction, deltadeltaG(int), obtained from double mutant cycle analysis showed that deltadeltaG(int) for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large deltadeltaG values are analyzed.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

Development and Optimization of Therapeutic Analogues of Anti-TNFα Antibody Infliximab

Previously, we have reported the crystal structures of Fab fragment of Infliximab in complex with TNFα. The structurally identified epitope on TNFα revealed the mechanism of TNFα inhibition by partially overlapping with the TNFα-receptor interface and the possibility to optimize the binding affinity. In this study, we launched a screen of a phage display library to isolate novel anti-TNFα antibodies based on the infliximab epitope. To develop novel anti-TNFα antibodies, structural analysis, the phage display antibody isolation, step by step antibody optimization, CDR residues random mutagenesis, and binding affinity characterization were performed. One of the novel antibodies generated on the backbone of infliximab, Inf3D6, has the superior binding affinity to TNFα, thus, demonstrating the potential for structure guided optimization for improvement of existing antibody-based therapeutics.     

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.     

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography

Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERRα in research and clinical applications.     

N-terminal flanking region of A1 domain in von Willebrand factor stabilizes structure of A1A2A3 complex and modulates platelet activation under shear stress

von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ibα. We have analyzed two A1A2A3 tri-domain proteins to demonstrate that the amino acid sequence, Gln(1238)-Glu(1260), in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIbα binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln(1238)-Glu(1260) stabilizes the structural conformation of the A1A2A3 tri-domain complex. The structural stabilization imparted by this particular region inhibits the binding capacity of the tri-domain protein for GPIbα. Deletion of this region causes a conformational change in the A1 domain that increases binding to GPIbα. Only the truncated protein was capable of effectively blocking ristocetin-induced platelet agglutination. To determine the capacity of activating platelets via the interaction with GPIbα, whole blood was incubated with the N-terminal region truncated or intact tri-A domain protein prior to perfusion over a fibrin(ogen)-coated surface. At a high shear rate of 1,500 s(-1), platelets from blood containing the truncated protein rapidly bound, covering >90% of the fibrin(ogen) surface area, whereas the intact tri-A domain protein induced platelets to bind <10%. The results obtained in this study ascertain the relevant role of the structural association between the N-terminal flanking region of the A1 domain (amino acids Gln(1238)-Glu(1260)) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIbα in solution under high shear forces.     

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination +

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.     

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: Application to antibodies against epidermal growth factor

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.     

Molecular dynamics simulation and steered molecular dynamics simulation on irisin dimers

Irisin is found closely associated with promoting the browning of beige fat cells in white adipose tissue. The crystal structure reveals that irisin forms a continuous inter-subunit β-sheet dimer. Here, molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation were performed to investigate the dissociation process and the intricate interactions between the two irisin monomers. In the process of MD, the interactions between the monomers were roughly analyzed through the average numbers of both hydrophobic contacts and H-bonds. Then, SMD was performed to investigate the accurate interaction energy between the monomers. By the analysis of dissociation energy, the van der Waals (vdW) force was identified as the major energy to maintain the dimer structure, which also verified the results of MD simulation. Meanwhile, 11 essential residues were discovered by the magnitude of rupture force during dissociation. Among them, residues Arg75, Glu79, Ile77, Ala88, and Trp90 were reported in a previous study using the method of mutagenesis and size exclusion chromatography, and several new important residues (Arg72, Leu74, Phe76, Gln78, Val80, and Asp91) were also identified. Interestingly, the new important residues that we discovered and the important residues that were reported are located in the opposite side of the β-sheet of the dimer.     

Structure of IL-17A in Complex with a Potent, Fully Human Neutralizing Antibody

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 Å resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.     

Agkistin, a snake venom-derived glycoprotein Ib antagonist, disrupts von Willebrand factor-endothelial cell interaction and inhibits angiogenesis

Glycoprotein (GP) Ib, an adhesion receptor expressed on both platelets and endothelial cells, mediates the binding of von Willebrand factor (vWF). Platelet GPIb plays an important role in platelet adhesion and activation, whereas the interaction of vWF and endothelial GPIb is not fully understood. We report here that agkistin, a snake venom protein, selectively blocks the interaction of vWF with human endothelial GPIb and inhibits angiogenesis in vivo. Agkistin specifically blocked human umbilical vein endothelial cell (HUVEC) adhesion to immobilized vWF in a concentration-dependent manner. Fluorescein isothiocyanate (FITC)-conjugated agkistin bound to HUVECs in a saturable manner. AP1, a monoclonal antibody (mAb) raised against GPIb, specifically inhibited the binding of FITC-conjugated agkistin to HUVECs in a dose-dependent manner, but other anti-integrin mAbs raised against alpha(v)beta(3), alpha(2)beta(1), and alpha(5)beta(1) did not affect this binding reaction. However, neither agkistin (2 microgram/ml) nor AP1 (40 microgram/ml) apparently reduced HUVEC viability. Both agkistin and AP1 exhibited a profound anti-angiogenic effect in vivo when assayed by using the 10-day-old embryo chick chorioallantoic membrane model. These results suggest endothelial GPIb plays a role in spontaneous angiogenesis in vivo, and the anti-angiogenic effect of agkistin may be because of disruption of the interaction of endogenous vWF with endothelial GPIb.     

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.     

Molecular basis of the recognition of arachidonic acid by cytochrome P450 2E1 along major access tunnel

Cytochrome P450 2E1 is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids (e.g., arachidonic acid (AA)). In this study, we investigated the structural features of the AA‐bound CYP2E1 complex utilizing molecular dynamics (MD) and found that the distinct binding modes for both AA and fatty acid analog are conserved. Moreover, multiple random acceleration MD simulations and steered MD simulations uncovered the most possible tunnel for fatty acids. The main attractions are derived from three key residues, His107, Ala108, and His109, whose side chains reorient to keep ligands bound via hydrogen bonds during the initial unbinding process. More importantly, based on the calculated binding free energy results, we hypothesize that the hydrogen bonds between the receptor and the ligand are the most important contributors involved in the binding affinity. Thus, it is inferred that the hydrogen bonds between these three residues and the ligand may help offer insights into the structural basis of the different ligand egress mechanisms for fatty acids and small weight compounds. Our investigation provides detailed atomistic insights into the structural features of human CYP2E1–fatty acid complex structures. Furthermore, the ligand‐binding characteristics obtained in the present study are helpful for both experimental and computational studies of CYPs and may allow future researchers to achieve desirable changes in enzymatic activities. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 53–66, 2015.     

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIbα in platelet-type von Willebrand disease

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ibα (GPIbga), a major component of the platelet (GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPIα protein containing residues 228–238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a β-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic ‘face’ consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphiphatic surfaces in the 228–238 region of the Val 233-containing GPIbα protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.     

白藜芦醇苷对血栓形成及血浆TXB2和6-keto-PGF1α水平的影响

研究了白藜芦醇苷(polydatim,PD)的抗血栓形成作用及其作用机制。采用小鼠尾静脉注射花生四烯酸(arachidonicacid,AA)、电刺激大鼠颈动脉血栓形成方法评价PD的抗血栓形成作用;运用放射免疫法测定polydatin对兔血浆血栓素B2(thromboxaneB2,TXB2)及6酮前列腺素F1α(6ketoprostaglandinF1α,6ketoPGF1α)水平的影响。结果显示PD对AA、电刺激大鼠颈动脉引起的血栓形成具有明显的对抗作用;PD亦能降低兔血浆TXB2含量并升高6KetoPGF1α水平。本实验提示PD有明显的抗血栓形成作用,其机制可能与其降低血浆TXB2含量及升高6KetoPGF1α水平密切相关。     

A deeper analysis of the epitope/paratope of PLY-5, a mouse monoclonal antibody which recognises the conserved undecapeptide tryptophan-rich loop (ECTGLAWEWWR) of bacterial cholesterol-dependent cytolysins

A previous study showed that the minimal epitope recognised by the PLY-5 mAb in the conserved undecapeptide Trp-rich loop of bacterial CDCs should consist of WEWWRT (Jacobs et al., 1999) [5]. Now, through immunoscreening of amino acid substitution analogues, it is concluded that the second Trp and the Arg residues are essential in the PLY-5 epitope. The E residue is an auxiliary epitope contributor. Antibody modelling and docking simulations provided support for these findings. For recognition by the antibody, the Trp-rich loop flipped out, mimicking the mechanism of membrane insertion. The displaced second Trp was seen to establish aromatic stacking interactions with aromatic residues of the antibody paratope and the notably extruded guanidium tip of the arginine residue mediated electrostatic interactions with well-exposed carboxylic groups of glutamic residues on the surface of the paratope. Thus, the epitope/paratope interaction is mainly mediated by aromatic and by ionic interactions.     

Steered molecular dynamics simulations on the "tail helix latch" hypothesis in the gelsolin activation process

The molecular basis of the "tail helix latch" hypothesis in the gelsolin activation process has been studied by using the steered molecular dynamics simulations. In the present nanosecond scale simulations, the tail helix of gelsolin was pulled away from the S2 binding surface, and the required forces were calculated, from which the properties of binding between the tail helix and S2 domain and their dynamic unbinding processes were obtained. The force profile provides a detailed rupture mechanism that includes six major unbinding steps. In particular, the hydrogen bonds formed between Arg-207 and Asp-744 and between Arg-221 and Leu-753 are of the most important interaction pairs. The two hydrogen bond "clamps" stabilize the complex. The subsequent simulation on Arg-207-Ala (R207A) mutation of gelsolin indicated that this mutation facilitates the unbinding of the tail helix and that the contribution of the hydrogen bond between Arg-207 and Asp-744 to the binding is more than 50%, which offers a new clue for further mutagenesis study on the activation mechanism of gelsolin. Surrounding water molecules enhance the stability of the tail helix and facilitate the rupture process. Additionally, temperature also has a significant effect on the conformation of the arginine and arginine-related interactions, which revealed the molecular basis of the temperature dependence in gelsolin activation.