Protein tyrosine phosphatase 1B targets focal adhesion kinase and paxillin in cell-matrix adhesions.

Protein tyrosine phosphatase 1B (PTP1B, also known as PTPN1) is an established regulator of cell-matrix adhesion and motility. However, the nature of substrate targets at adhesion sites remains to be validated. Here, we used bimolecular fluorescence complementation assays, in combination with a substrate trapping mutant of PTP1B, to directly examine whether relevant phosphotyrosines on paxillin and focal adhesion kinase (FAK, also known as PTK2) are substrates of the phosphatase in the context of cell-matrix adhesion sites. We found that the formation of catalytic complexes at cell-matrix adhesions requires intact tyrosine residues Y31 and Y118 on paxillin, and the localization of FAK at adhesion sites. Additionally, we found that PTP1B specifically targets Y925 on the focal adhesion targeting (FAT) domain of FAK at adhesion sites. Electrostatic analysis indicated that dephosphorylation of this residue promotes the closed conformation of the FAT 4-helix bundle and its interaction with paxillin at adhesion sites.

Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer.

The underpinnings of STAT3 hyperphosphorylation resulting in enhanced signaling and cancer progression are incompletely understood. Loss-of-function mutations of enzymes that dephosphorylate STAT3, such as receptor protein tyrosine phosphatases, which are encoded by the PTPR gene family, represent a plausible mechanism of STAT3 hyperactivation. We analyzed whole exome sequencing (n = 374) and reverse-phase protein array data (n = 212) from head and neck squamous cell carcinomas (HNSCCs). PTPR mutations are most common and are associated with significantly increased phospho-STAT3 expression in HNSCC tumors. Expression of receptor-like protein tyrosine phosphatase T (PTPRT) mutant proteins induces STAT3 phosphorylation and cell survival, consistent with a "driver" phenotype. Computational modeling reveals functional consequences of PTPRT mutations on phospho-tyrosine-substrate interactions. A high mutation rate (30%) of PTPRs was found in HNSCC and 14 other solid tumors, suggesting that PTPR alterations, in particular PTPRT mutations, may define a subset of patients where STAT3 pathway inhibitors hold particular promise as effective therapeutic agents.

Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

ATPase subdomain IA is a mediator of interdomain allostery in Hsp70 molecular chaperones.

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the ß-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.

Convergence and equilibrium in molecular dynamics simulations.

Molecular dynamics is a powerful tool that has been long used for the simulation of biomolecules. It complements experiments, by providing detailed information about individual atomic motions. But there is an essential and often overlooked assumption that, left unchecked, could invalidate any results from it: is the simulated trajectory long enough, so that the system has reached thermodynamic equilibrium, and the measured properties are converged? Previous studies showed mixed results in relation to this assumption. This has profound implications, as the resulting simulated trajectories may not be reliable in predicting equilibrium properties. Yet, this is precisely what most molecular dynamics studies do. So the question arises: are these studies even valid?Here, we present a thorough analysis of up to a hundred microseconds long trajectories, of several system with varying size, to probe the convergence of different structural, dynamical and cumulative properties, and elaborate on the relevance of the concept of equilibrium, and its physical and biological meaning. The results show that properties with the most biological interest tend to converge in multi-microsecond trajectories, although other properties-like transition rates to low probability conformations-may require more time.

Phosphate uptake in PhoX: Molecular mechanisms.

PhoX is a high-affinity phosphate binding protein, present in Xanthomonas citri, a phytopathogen responsible for the citrus canker disease. Performing molecular dynamics simulations and different types of computational analyses, we study the molecular mechanisms at play in relation to phosphate binding, revealing the global functioning of the protein: PhoX naturally oscillates along its global normal modes, which allow it to explore both bound and unbound conformations, eventually attracting a nearby negative phosphate ion to the highly positive electrostatic potential on its surface, particularly close to the binding pocket. There, several hydrogen bonds are formed with the two main domains of the structure. Phosphate creates, in this way, a strong bridge that connects the domains, keeping itself between them, in a tight closed conformation, explaining its high binding affinity.

NADPH oxidase 2 activity disrupts Calmodulin/CaMKIIα complex via redox modifications of CaMKIIα-contained Cys30 and Cys289: Implications in Parkinson's disease.

Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) signaling in the brain plays a critical role in regulating neuronal Ca2+ homeostasis. Its dysfunctional activity is associated with various neurological and neurodegenerative disorders, including Parkinson's disease (PD). Using computational modeling analysis, we predicted that, two essential cysteine residues contained in CaMKIIα, Cys30 and Cys289, may undergo redox modifications impacting the proper functioning of the CaMKIIα docking site for Ca2+/CaM, thus impeding the formation of the CaMKIIα:Ca2+/CaM complex, essential for a proper modulation of CaMKIIα kinase activity. Our subsequent in vitro investigations confirmed the computational predictions, specifically implicating Cys30 and Cys289 residues in impairing CaMKIIα:Ca2+/CaM interaction. We observed CaMKIIα:Ca2+/CaM complex disruption in dopamine (DA) nigrostriatal neurons of post-mortem Parkinson's disease (PD) patients' specimens, addressing the high relevance of this event in the disease. CaMKIIα:Ca2+/CaM complex disruption was also observed in both in vitro and in vivo rotenone models of PD, where this phenomenon was associated with CaMKIIα kinase hyperactivity. Moreover, we observed that, NADPH oxidase 2 (NOX2), a major enzymatic generator of superoxide anion (O2●-) and hydrogen peroxide (H2O2) in the brain with implications in PD pathogenesis, is responsible for CaMKIIα:Ca2+/CaM complex disruption associated to a stable Ca2+CAM-independent CaMKIIα kinase activity and intracellular Ca2+ accumulation. The present study highlights the importance of oxidative stress, in disturbing the delicate balance of CaMKIIα signaling in calcium dysregulation, offering novel insights into PD pathogenesis.

Relative stability of the open and closed conformations of the active site loop of streptavidin.

The eight-residue surface loop, 45-52 (Ser, Ala, Val, Gly, Asn, Ala, Glu, Ser), of the homotetrameric protein streptavidin has a "closed" conformation in the streptavidin-biotin complex, where the corresponding binding affinity is one of the strongest found in nature (ΔG ∼ -18 kcal∕mol). However, in most of the crystal structures of apo (unbound) streptavidin, the loop conformation is "open" and typically exhibits partial disorder and high B-factors. Thus, it is plausible to assume that the loop structure is changed from open to closed upon binding of biotin, and the corresponding difference in free energy, ΔF = F(open) - F(closed) in the unbound protein, should therefore be considered in the total absolute free energy of binding. ΔF (which has generally been neglected) is calculated here using our "hypothetical scanning molecular-dynamics" (HSMD) method. We use a protein model in which only the atoms closest to the loop are considered (the "template") and they are fixed in the x-ray coordinates of the free protein; the x-ray conformation of the closed loop is attached to the same (unbound) template and both systems are capped with the same sphere of TIP3P water. Using the force field of the assisted model building with energy refinement (AMBER), we carry out two separate MD simulations (at temperature T = 300 K), starting from the open and closed conformations, where only the atoms of the loop and water are allowed to move (the template-water and template-loop interactions are considered). The absolute F(open) and F(closed) (of loop + water) are calculated from these trajectories, where the loop and water contributions are obtained by HSMD and a thermodynamic integration (TI) process, respectively. The combined HSMD-TI procedure leads to total (loop + water) ΔF = -27.1 ± 2.0 kcal∕mol, where the entropy TΔS constitutes 34% of ΔF, meaning that the effect of S is significant and should not be ignored. Also, ΔS is positive, in accord with the high flexibility of the open loop observed in crystal structures, while the energy ΔE is unexpectedly negative, thus also adding to the stability of the open loop. The loop and the 250 capped water molecules are the largest system studied thus far, which constitutes a test for the efficiency of HSMD-TI; this efficiency and technical issues related to the implementation of the method are also discussed. Finally, the result for ΔF is a prediction that will be considered in the calculation of the absolute free energy of binding of biotin to streptavidin, which constitutes our next project.

Physical interactions driving the activation/inhibition of calcium/calmodulin dependent protein kinase II.

CaMKII is a protein kinase whose function is regulated by the binding of the Calcium/Calmodulin complex (Ca2+/CaM). It is a major player in the Long Term Potentiation process where it acts as a molecular switch, oscillating between inhibited and active conformations. The mechanism for the switching is thought to be initiated by Ca2+/CaM binding, which allows the trans-phosphorylation of a subunit of CaMKII by a neighboring kinase, leading to the active state of the system. A combination of all-atom and coarse-grained MD simulations with free energy calculations, led us to reveal an interplay of electrostatic forces exerted by Ca2+/CaM on CaMKII, which initiate the activation process. The highly electrically charged Ca2+/CaM neutralizes basic regions in the linker domain of CaMKII, facilitating its opening and consequent activation. The emerging picture of CaMKII's behavior highlights the preponderance of electrostatic interactions, which are modulated by the presence of Ca2+/CaM and the phosphorylation of key sites.

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake.

Tumor extracellular matrix (ECM) is a high-capacity target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here, we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity, and specificity for precision targeting of the tumor ECM and malignant cells.

Sodium perchlorate effects on the helical stability of a mainly alanine peptide.

Sodium perchlorate salt (NaClO(4)) is commonly used as an internal intensity standard in ultraviolet resonance Raman (UVRR) spectroscopy experiments. It is well known that NaClO(4) can have profound effects on peptide stability. The impact of NaClO(4) on protein stability in UVRR experiments has not yet been fully investigated. It is well known from experiment that protein stability is strongly affected by the solution composition (water, salts, osmolytes, etc.). Therefore, it is of the utmost importance to understand the physical basis on which the presence of salts and osmolytes in the solution impact protein structure and stability. The aim of this study is to investigate the effects of NaClO(4), on the helical stability of an alanine peptide in water. Based upon replica-exchange molecular dynamics data, it was found that NaClO(4) solution strongly stabilizes the helical state and that the number of pure helical conformations found at room temperature is greater than in pure water. A thorough investigation of the anion effects on the first and second solvation shells of the peptide, along with the Kirkwood-Buff theory for solutions, allows us to explain the physical mechanisms involved in the observed specific ion effects. A direct mechanism was found in which ClO(4)(-) ions are strongly attracted to the folded backbone.

Structure of aqueous sodium perchlorate solutions.

Salt solutions have been the object of study of many scientists through history, but one of the most important findings came along when the Hofmeister series were discovered. Their importance arises from the fact that they influence the relative solubility of proteins, and solubility is directly related to one of today's holy grails: protein folding. In this work we characterize one of the more-destabilizing salts in the series, sodium perchlorate, by studying it as an aqueous solution at various concentrations ranging from 0.08 to 1.60 mol/L. Molecular dynamics simulations at room temperature permitted a detailed study of the organization of solvent and cosolvent, in terms of its radial distribution functions, along with the study of the structure of hydrogen bonds in the ions' solvation shells. We found that the distribution functions have some variations in their shape as concentration changes, but the position of their peaks is mostly unaffected. Regarding water, the most salient fact is the noticeable (although small) change in the second hydration shell and even beyond, especially for g(O(w)***O(w)), showing that the locality of salt effects should not be restricted to considerations of only the first solvation shell. The perturbation of the second shell also appears in the study of the HB network, where the difference between the number of HBs around a water molecule and around the Na(+) cation gets much smaller as one goes from the first to the second solvation shell, yet the difference is not negligible. Nevertheless, the effect of the ions past their first hydration shell is not enough to make a noticeable change in the global HB network. The Kirkwood-Buff theory of liquids was applied to our system, in order to calculate the activity derivative of the cosolvent. This coefficient, along with a previously calculated preferential binding, allowed us to establish that if a folded AP peptide is immersed in the studied solution, becoming the solute, then increasing the salt concentration will make the helix more stable.

Principle of maximum caliber and quantum physics.

MaxCal is a variational principle that can be used to infer distributions of paths in the phase space of dynamical systems. It has been successfully applied to different areas of classical physics, in particular statistical mechanics in and out of equilibrium. In this work, guided by the analogy of the formalism of MaxCal with that of the path integral formulation of quantum mechanics, we explore the extension of its applications to the realm of quantum physics, and show how the Lagrangians of both relativistic and nonrelativistic quantum fields can be built from MaxCal, with a suitable set of constraints. Related, the details of the constraints allow us to reinterpret the concept of inertia.

Mechanisms of Activation and Subunit Release in Ca2+/Calmodulin-Dependent Protein Kinase II.

Calcium/calmodulin-dependent protein kinase II is an enzyme involved in many different functions, including the so-called long-term potentiation, a mechanism that strengthens synapses in a persistent mode and is believed to be a basic cellular mechanism for memory formation. Here we study the conformational changes of the enzyme due to phosphorylation of some key residues that are believed to drive the transition from an inhibited to an active state; it is this active state the one associated with long-term potentiation. We found that the conformational changes could be explained in terms of three charged regions in the three main subdomains of the enzyme: the hub, linker, and kinase. The role of phosphorylation is to change the charge relation between them, turning on and off their interactions and switching between an attractive state (nonphosphorylated or inhibited) and a not attractive one (phosphorylated or active). We also show that phosphorylated subunits become less stable, and this could favor their release from the multimer, as has been already observed experimentally.

Dynamic Modulation of Binding Affinity as a Mechanism for Regulating Interferon Signaling.

How structural dynamics affects cytokine signaling is under debate. Here, we investigated the dynamics of the type I interferon (IFN) receptor, IFNAR1, and its effect on signaling upon binding IFN and IFNAR2 using a combination of structure-based mechanistic studies, in situ binding, and gene induction assays. Our study reveals that IFNAR1 flexibility modulates ligand-binding affinity, which, in turn, regulates biological signaling. We identified the hinge sites and key interactions implicated in IFNAR1 inter-subdomain (SD1-SD4) movements. We showed that the predicted cooperative movements are essential to accommodate intermolecular interactions. Engineered disulfide bridges, computationally predicted to interfere with IFNAR1 dynamics, were experimentally confirmed. Notably, introducing disulfide bonds between subdomains SD2 and SD3 modulated IFN binding and activity in accordance with the relative attenuation of cooperative movements with varying distance from the hinge center, whereas locking the SD3-SD4 interface flexibility in favor of an extended conformer increased activity.

Structure and Dynamics Study of LeuT Using the Markov State Model and Perturbation Response Scanning Reveals Distinct Ion Induced Conformational States.

The bacterial leucine transporter (LeuT), a close homologue of the eukaryote monoamine transporters (MATs), currently serves as a powerful template for computer simulations of MATs. Transport of the amino acid leucine through the membrane is made possible by the sodium electrochemical potential. Recent reports indicate that the substrate transport mechanism is based on structural changes such as hinge movements of key transmembrane domains. In order to further investigate the role of sodium ions in the uptake of leucine, here we present a Markov state model analysis of atomistic simulations of lipid embedded LeuT in different environments, generated by varying the presence of binding pocket sodium ions and substrate. Six metastable conformations are found, and structural differences between them along with transition probabilities are determined. We complete the analysis with the implementation of perturbation response scanning on our system, determining the most sensitive and influential regions of LeuT, in each environment. Our results show that the occupation of sites Na1 and Na2, along with the presence of the substrate, selectively influences the geometry of LeuT. In particular, the occupation of each site Na1/Na2 has strong effects (in terms of changes in influence and/or sensitivity, as compared to the case without ions) in specific regions of LeuT, and the effects are different for simultaneous occupation. Our results strengthen the rationale and provide a conformational mechanism for a putative transport mechanism in which Na2 is necessary, but may not be sufficient, to initiate and stabilize extracellular substrate access to the binding pocket.

Absolute Free Energy of Binding and Entropy of the FKBP12-FK506 Complex: Effects of the Force Field.

The hypothetical scanning molecular dynamics (HSMD) method combined with thermodynamic integration (HSMD-TI) has been extended recently for calculating ΔA(0)-the absolute free energy of binding of a ligand to a protein. With HSMD-TI, ΔA(0) is obtained in a new way as a sum of several components, among them is ΔSligand-the change in the conformational entropy as the ligand is transferred from the bulk solvent to the active site-this entropy is obtained by a specific reconstruction procedure. This unique aspect of HSMD (which is useful in rational drug design) is in particular important for treating large ligands, where ΔSligand might be significant. Technically, one should verify that the results for ΔSligand converge-a property that might become more difficult for large ligands; therefore, studying ligands of increasing size would define the range of applicability of HSMD-TI for binding. In this paper, we check the performance of HSMD-TI by applying it to the relatively large ligand FK506 (126 atoms) complexed with the protein FKBP12, where ΔA(0) = -12.8 kcal/mol is known experimentally as well as the crystal structure of the complex. This structure was initially equilibrated by carrying out a 100 ns molecular dynamics trajectory, where the system is modeled by the AMBER force field, TIP3P water, and Particle Mesh Ewald. HSMD-TI calculations were carried out in three conformational regions defined by the intervals [0.2,2], [2,5], and [5,100] ns along the trajectory, where local equilibration of the total energy has been observed; we obtained ΔA(0) = -13.6 ± 1.1, -16.6 ± 1.4, and -16.7 ± 1.4 kcal/mol, respectively indicating the following: (1) The second and third regions belong to the same conformational subspace of the complex, which is different from the [0.2,2] ns subspace. (2) The unsatisfactory result for ΔA(0) obtained in the well equilibrated (hence theoretically preferred) latter regions reflects the nonperfect modeling used, which however (3) has led to the experimental ΔA(0) in the [0.2,2] ns region close to the crystal structure. Keeping the complex near its crystal structure has been a successful approach in the literature. To check this avenue further, we applied harmonic restraints on backbone atoms and obtained unsatisfactory results for ΔA(0), suggesting that implementation of this approach is not straightforward. Converging results for ΔSligand were obtained in all regions, where the result ΔSligand([0.2,2]) = 7.1 ± 1.2 kcal/mol is less region dependent than ΔA(0) and is relatively large probably due to the large ligand.

Absolute free energy of binding of avidin/biotin, revisited.

The binding of biotin to avidin is one of the strongest in nature with absolute free energy of binding, ΔA(0) = -20.4 kcal/mol. Therefore, this complex became a target for a large number of computational studies, which all, however, are based on approximate techniques or simplified models and have led to a wide range of results Therefore, ΔA(0) is calculated here by rigorous statistical mechanical methods and models that consider long-range electrostatics. (1) We apply our method, "hypothetical scanning molecular dynamics with thermodynamic integration" (HSMD-TI) to avidin-biotin modeled by periodic boundary conditions with particle mesh ewald (PME). (2) We apply the double decoupling method (DDM) to this system modeled by the spherical solvent boundary potential (SSBP) and the generalized solvent boundary potential (GSBP). The corresponding results for neutral biotin, ΔA(0) = -29.1 ± 0.8 and -25.2 ± 0.5 kcal/mol are significantly lower than the experimental value; we also provide the result for a charged biotin, ΔA(0) = -33.3 ± 0.8 kcal/mol. It is plausible to suggest that this disagreement with the experiment may stem from ignoring the (positive) contribution of a mobile loop that changes its structure upon ligand binding.

Calculation of the Absolute Free Energy of Binding and Related Entropies with the HSMD-TI Method: The FKBP12-L8 Complex.

The hypothetical scanning molecular dynamics (HSMD) method is used here for calculating the absolute free energy of binding, ΔA(0) of the complex of the protein FKBP12 with the ligand SB2 (also denoted L8) - a system that has been studied previously for comparing the performance of different methods. Our preliminary study suggests that considering long-range electrostatics is imperative even for a hydrophobic ligand such as L8. Therefore the system is modeled by the AMBER force field using Particle Mesh Ewald (PME). HSMD consists of three stages applied to both the ligand-solvent and ligand-protein systems. (1) A small set of system configurations (frames) is extracted from an MD trajectory. (2) The entropy of the ligand in each frame is calculated by a reconstruction procedure. (3) The contribution of water and protein to ΔA(0) is calculated for each frame by gradually increasing the ligand-environment interactions from zero to their full value using thermodynamic integration (TI). Unlike the conventional methods, the structure of the ligand is kept fixed during TI, and HSMD is thus free from the end-point problem encountered with the double annihilation method (DAM); therefore, the need for applying restraints is avoided. Furthermore, unlike the conventional methods, the entropy of the ligand and water is obtained directly as a byproduct of the simulation. In this paper, in addition to the difference in the internal entropies of the ligand in the two environments, we calculate for the first time the external entropy of the ligand, which provides a measure for the size of the active site. We obtain ΔA(0) = -10.7 ±1.0 as compared to the experimental values -10.9 and -10.6 kcal/mol. However, a protein/water system treated by periodic boundary conditions grows significantly with increasing protein size and the computation of ΔA(0) would become expensive by all methods. Therefore, we also apply HSMD to FKBP12-L8 described by the GSBP/SSBP model of Roux's group (implemented in the software CHARMM) where only part of the protein and water around the active site are considered and long-range electrostatic effects are taken into account. For comparison this model was also treated by the double decoupling method (DDM). The two methods have led to comparable results for ΔA(0) which are somewhat lower than the experimental value. The ligand was found to be more confined in the active site described by GSBP/SSBP than by PME where its entropy in solvent is larger than in the active site by 1.7 and by 5.5 kcal/mol, respectively.

New method for calculating the absolute free energy of binding: the effect of a mobile loop on the avidin/biotin complex.

Hypothetical scanning molecular dynamics (HSMD) is a relatively new method for calculating the absolute free energy and entropy. HSMD is extended here for the first time for calculating the absolute free energy of binding, ΔA(0), as applied to the avidin-biotin complex. With HSMD the ligand is built (more accurately reconstructed) from nothing in solvent and in the protein, in contrast to the commonly used methods where the ligand is annihilated (by thermodynamic integration) in these environments. Therefore, the end-point problem encountered with the latter methods does not exist with HSMD and the need for restraints is avoided. Also, the entropy of the ligand and water in both environments is obtained directly as a byproduct of the simulation. The binding mechanism of biotin to avidin involves a mobile loop that is expected to be in an open conformation in unbound avidin, which is changed to a closed one upon binding, that is, the loop moves to cover biotin in the active site. The contribution of the loop's conformational change to the total free energy of binding is calculated here for the first time. Our result, ΔA(0) = -24.9 ± 7 covers the experimental value -20.7 kcal/mol within the error bars.