Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: equilibrium, kinetic, and crystallographic studies.

Stringent specificity and complementarity between the receptor, a periplasmic phosphate-binding protein (PBP) with a two-domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Ala197, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP-phosphate complex retains wild-type Kd and X-ray structure beyond the replacement residue. The Kd (0.18 microM) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped-flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed-conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.     

Mechanisms of protein-ligand association and its modulation by protein mutations

Protein-ligand interactions are essential for nearly all biological processes, and yet the biophysical mechanism that enables potential binding partners to associate before specific binding occurs remains poorly understood. Fundamental questions include which factors influence the formation of protein-ligand encounter complexes, and whether designated association pathways exist. To address these questions, we developed a computational approach to systematically analyze the complete ensemble of association pathways. Here, we use this approach to study the binding of a phosphate ion to the Escherichia coli phosphate-binding protein. Various mutants of the protein are considered, and their effects on binding free-energy profiles, association rates, and association pathway distributions are quantified. The results reveal the existence of two anion attractors, i.e., regions that initially attract negatively charged particles and allow them to be efficiently screened for phosphate, which is subsequently specifically bound. Point mutations that affect the charge on these attractors modulate their attraction strength and speed up association to a factor of 10 of the diffusion limit, and thus change the association pathways of the phosphate ligand. It is demonstrated that a phosphate that prebinds to such an attractor neutralizes its attraction effect to the environment, making the simultaneous association of a second phosphate ion unlikely. This study suggests ways in which structural properties can be used to tune molecular association kinetics so as to optimize the efficiency of binding, and highlights the importance of kinetic properties.     

Investigating a back door mechanism of actin phosphate release by steered molecular dynamics

In actin-based cell motility, phosphate (Pi) release after ATP hydrolysis is an essential biochemical process, but the actual pathway of Pi separation from actin is not well understood. We report a series of molecular dynamics simulations that induce the dissociation of Pi from actin. After cleavage from ATP, the singly protonated phosphate (HPO4(2-)) rotates about the ADP-associated Ca2+ ion, turning away from the negatively charged ADP towards the putative exit near His73. To reveal the microscopic processes underlying the release of Pi, adhesion forces were measured when pulling the substrate out of its binding pocket. The results suggest that the separation from the divalent cation is the rate-limiting step in Pi release. Protonation of HPO4(2-) to H2PO4- lowers the electrostatic barrier during Pi liberation from the ion. The simulations revealed a propensity of charged His73+ to form a salt bridge with HPO4(2-), but not with H2PO4-. His73 stabilizes HPO4(2-) and, thereby, inhibits rapid Pi release from actin. Arg177 remains attached to Pi along the putative back door pathway, suggesting a shuttle function that facilitates the transport of Pi to a binding site on the protein surface.     

A Brownian dynamics study: the effect of a membrane environment on an electron transfer system

During the past few years, three-dimensional crystal structures of many of the important integral membrane proteins responsible for the bioenergetic processes of photosynthesis and respiration have been determined. Moreover, a few crystal structures of protein-protein complexes have become available that characterize the interaction between those membrane proteins and the electron carrier protein cytochrome c. Here, we address the association kinetics for binding of cytochrome c to cytochrome c oxidase (COX) from Paracoccus denitrificans by Brownian dynamics simulations. The effects of ionic strength and protein mutations were studied for two different cytochrome c species: the positively charged, dipolar horse heart cytochrome c and the negatively charged physiological electron transfer partner cytochrome c(552). We studied association toward "naked" COX and toward membrane-embedded COX where the membrane is represented as an uncharged DPPC bilayer modeled in atomistic detail. For the nonnatural association toward "naked" COX, the association rates are >100 times larger for horse heart cytochrome c than for cytochrome c(552). Interestingly, the presence of the lipid bilayer leads to a dramatic decrease of the association rate of horse heart cytochrome c, but slightly enhances association of cytochrome c(552), leading to very similar association rates of both proteins to membrane-embedded COX. This finding from computational modeling studies may reflect the optimization of surface patches and of the total net charge on electron transfer pairs in nature.     

The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C

Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold. The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known. Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin. To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C.     

含磷酸结合口袋的BRCT结构域功能位点预测

BRCT( BRCA1 C-terminus)是真核生物DNA损伤修复系统重要的信号传导和蛋白靶向结构域.为了探讨含磷酸结合口袋的BRCT与磷酸化配体结合的机制,对XRCC1 BRCT1、PTIP BRCT4、ECT2 BRCT1和TopBP1BRCT1进行了结构保守性和表面静电势分析.结果显示,4个BRCT的磷酸结合口袋周围所存在的结构保守并带正电势的沟槽很可能是其功能位点,并且类似的沟槽在含磷酸结合口袋的BRCT中普遍存在.沟槽两侧及底部均带有极性氨基酸残基,两侧带正电荷,而底部疏水.这说明沟槽与配体的结合以静电和疏水相互作用为主.沟槽主要位于单个BRCT中,而且4个BRCT的沟槽在形状和电荷分布上都不同,说确明BRCT配体特异性主要由单个BRCT决定.磷酸结合口袋位于沟槽中心,说明沟槽可能同时结合磷酸化残基的N端和C端附近残基.     

PhoE protein pores in the outer membrane of Escherichia coli K-12 not only have a preference for Pi and Pi-containing solutes but are general anion-preferring channels

Previous studies on the question of whether the PhoE protein pore has a preference for P_i and P_i-containing solutes only or whether it constitutes a general anion-preferring channel, have not given an unequivocal answer either because the presence of the phosphate binding protein was not ascertained or because only arsenate was tested as a non P_i-containing control solute. Permeability properties of PhoE, OmpF and OmpC protein pores for negatively charged solutes were measured in vivo in the presence of phosphate-binding protein. It appeared that the PhoE protein pore is the most efficient channel for the three tested solutes phosphate, succinate and sulphate. Conditions were established to measure the frequency of ethyl methane sulphonate induced mutations as a function of the presence of pore proteins. These results indicate that PhoE protein also forms the most efficient channel for ethyl methane sulphonate. We conclude that the preference of the PhoE protein pore is not restricted to P_i and P_i-containing solutes but also concerns several other negatively charged solutes.     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

A synthetic hexapeptide designed to resemble a proteinaceous P-loop nest is shown to bind inorganic phosphate

The hexapeptide Ser-Gly-Ala-Gly-Lys-Thr has been synthesized and characterized. It was designed as a minimal soluble peptide that would be likely to have the phosphate-binding properties observed in the P-loops of proteins that bind the β-phosphate of GTP or ATP. The β-phosphate in such proteins is bound by a combination of the side chain ε-amino group of the lysine residue plus the concavity formed by successive main chain peptide NH groups called a nest, which is favored by the glycines. The hexapeptide is shown to bind HPO(4) (2-) strongly at neutral pH. The affinities of the various ionized species of phosphate and hexapeptide are analyzed, showing that they increase with pH. It is likely the main chain NH groups of the hexapeptide bind phosphate in much the same way as the corresponding P-loop atoms bind the phosphate ligand in proteins. Most proteinaceous P-loops are situated at the N-termini of α-helices, and this observation has frequently been considered a key aspect of these binding sites. Such a hexapeptide in isolation seems unlikely to form an α-helix, an expectation in accord with the CD spectra examined; this suggests that being at the N-terminus of an α-helix is not essential for phosphate binding. An unexpected finding about the hexapeptide-HPO(4) (2-) complex is that the side chain ε-amino group of the lysine occurs in its deprotonated form, which appears to bind HPO(4) (2-) via an N···H-O-P hydrogen bond.     

Association of Cytochrome c with Membrane-Bound Cytochrome c Oxidase Proceeds Parallel to the Membrane Rather Than in Bulk Solution

Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c₅₅₂, and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.     

Restoration of phosphate transport by the phosphate-binding protein in spheroplasts of Escherichia coli.

Reconstitution of phosphate transport in Escherichia coli was demonstrated. Conversion of E. coli K10 cells to spheroplasts decreased phosphate transport to about 2%. Addition of purified phosphate-binding protein at physiological levels to these spheroplasts caused a mean 14-fold increase in phosphate transport rate. Crude shock fluid fractions were also stimulatory but not if the shock fluid was obtained from mutants lacking phosphate-binding protein. The effect of the binding protein was abolished by its specific antibody. The phosphate was shown to have entered the cell, where it became esterified. Reconstitution was not possible with cold-shocked or osmotically shocked cells.     

Controlling protein transport in ultrafiltration using small charged ligands

Previous studies have demonstrated that protein transport during ultrafiltration can be strongly influenced by solution pH and ionic strength. The objective of this study was to examine the possibility of controlling protein transmission using a small, highly charged ligand that selectively binds to the protein of interest. Experiments were performed using bovine serum albumin and the dye Cibacron Blue. Protein sieving data were obtained with essentially neutral and negatively charged versions of a composite regenerated cellulose membrane to examine the effects of electrostatic interactions. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand binding and the increase in solution ionic strength due to the free ligand in solution.     

A universal small molecule,inorganic phosphate,restricts the substrate specificity of Dicer-2 in small RNA biogenesis

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme.     

Thrombin inhibitor design: X-ray and solution studies provide a novel P1 determinant

The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was confirmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent channel beneath the S1 pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium concentration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor.     

Identification of optimal anion spacing for anti-HIV activity in a series of cosalane tetracarboxylates

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. An investigation of the optimal anion distances for anti-HIV activity in a series of cosalane tetracarboxylate analogues has been completed, and maximal activity results when the two proximal and the two distal carboxylates are separated by eight atoms.     

Decomposition analysis of free energy profile for Hsp90-ADP association

The functional cycle of heat shock protein 90 (Hsp90) is driven and inhibited by the association/dissociation of ligand molecules. In order to understand the molecular mechanism of the association of N-terminal domain of Hsp90 (N-Hsp90) and its ligand molecule, it is necessary to investigate which part in the target system promotes or inhibits the association of N-Hsp90 and its ligand molecule. We apply the decomposition analysis for the association free energy of N-Hsp90 and ADP. The mean force calculated by thermodynamic integration method combined with molecular dynamic simulations is divided into the contributions from molecules in the target system. Van der Waals interaction of the solvent water molecules strongly stabilises the association. Three lysine residues on the surface of the N-Hsp90 pull ADP toward the binding pocket of N-Hsp90. This study elucidates the association process of ADP from the bulk region to the binding pocket of the N-terminal domain Hsp90. This approach is applicable to elucidate the association process of biomolecules.     

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.     

Anti-HIV activity of a series of cosalane amino acid conjugates

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. The purpose of the present study was to examine the hypothesis that the two carboxyl groups of cosalane could be sacrificed through conjugation to amino acids, and the anti-HIV activity still be retained, provided that at least two new carboxyl groups are contributed by the amino acid residues.