Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.     

Distinct regions of bacterial flagellar switch protein FliM interact with FliG, FliN and CheY

The FliG, FliM, and FliN proteins of the bacterial flagellar motor are believed to interact with one another to form the switch complex, which in turn is thought to interact with one of the chemotaxis proteins, CheY. In particular, FliM appears to be an intermediary between CheY and FliG: the current model suggests that CheY, when phosphorylated (CheY-P), binds to FliM and produces a conformational change in FliM that is propagated to FliG. The result of these interactions is to induce clockwise rotation of the flagellar motors and tumbling of the cell. Various genetic and biochemical studies have provided evidence that the switch proteins associate with each other and that CheY-P binds to FliM. Here, we have used affinity blotting to obtain direct evidence of interaction between Salmonella typhimurium FliM and FliN, FliM and FliG, and FliM and CheY-P. We have also examined the ability of various FliM deletion and truncation mutant proteins to bind to FliN, FliG, and CheY-P. From these data, we conclude that distinct regions of the FliM protein bind to each of these other proteins. We propose a model in which the N-terminal region of FliM binds to CheY-P, the middle region of FliM binds to FliG, and the C-terminal region binds to FliN.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

The role of charged residues mediating low affinity protein-protein recognition at the cell surface by CD2

Insights into the structural basis of protein-protein recognition have come principally from the analysis of proteins such as antibodies, hormone receptors, and proteases that bind their ligands with relatively high affinity (Ka approximately 10(9) M-1). In contrast, few studies have been done on the very low affinity interactions mediating cell adhesion and cell-cell recognition. As a site of protein-protein recognition, the ligand binding face of the T lymphocyte cell-cell recognition molecule, CD2, which binds its ligands 10(4)- to 10(5)-fold more weakly than do antibodies and proteases, is unusual in being both very flat and highly charged. An analysis of the effect of mutations and ionic strength on CD2 binding to its ligand, CD48, indicates that these charged residues contribute little, if any, binding energy to this interaction. However, the loss of these charged residues is shown to markedly reduce ligand-binding specificity. Thus, the charged residues increase the specificity of CD2 binding without increasing the affinity. This phenomenon is likely to result from a requirement for electrostatic complementarity between charged binding surfaces to compensate for the removal, upon binding, of water interacting with the charged residues. It is proposed that this mode of recognition is highly suited to biological interactions requiring a low affinity because it uncouples increases in specificity from increases in affinity.     

Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis

High-affinity mu-conotoxin block of skeletal muscle Na+ channels depends on an arginine at position 13 (Arg-13). To understand both the mechanism of toxin interaction and the general structure of its binding site in the channel mouth, we examined by thermodynamic mutant cycle analysis the interaction between the critical Arg-13 and amino acid residues known to be in the channel's outer vestibule. Arg-13 interacts specifically with domain II Glu-758 with energy of about -3.0 kcal/mol, including both electrostatic and nonelectrostatic components, and with Glu-403 with energy of about -2.0 kcal/mol. Interactions with the other charged residues in the outer vestibule were shown to be almost entirely electrostatic, because these interactions were maintained when Arg-13 was replaced by lysine. These results place the bound Arg-13 at the channel mouth adjacent to the P (pore) loops of domains I and II. Distance estimates based on interaction energies suggest that the charged vestibule residues are in relative positions similar to those of the Lipkind-Fozzard vestibule model [Lipkind, G. M., and Fozzard, H. A. (1994) Biophys. J. 66, 1-13]. Kinetic analysis suggests that Arg-13 interactions are partially formed in the ligand-channel transition state.     

Modelling the three-dimensional structure and the electrostatic potential field of two Cu,Zn superoxide dismutase variants from tomato leaves

The three-dimensional structure of tomato P31 and T10 Cu,Zn superoxide dismutases (SODs) were computer modelled using the structure of the bovine enzyme as a template. The structure-essential residues retain in the models the position occupied in the other Cu,Zn SODs of known 3D structure and the overall packing of the beta-barrel is maintained. Formation of 'aromatic pairs' occurs between newly inserted aromatic residues. The number of total charges changes in the two variants and some charged residues located in the proximity of the active site in most Cu,Zn SODs disappear in tomato enzymes. Calculation of the electrostatic potential field, carried out by numerically solving the Poisson-Boltzmann equation, indicates that in both variants a negative potential field surrounds all the protein surface except the active site areas, characterized by positive potential values, as already observed in the bovine enzyme. This result confirms that coordinated mutations of charged residues have occurred in the evolution of this enzyme giving rise to a peculiar electrostatic potential distribution common to all members of this protein family.     

Hepatocyte attachment on biodegradable modified chitosan membranes: in vitro evaluation for the development of liver organoids

Electron transfer reactions involving protein-protein interactions require the formation of a transient complex which brings together the two redox centres exchanging electrons. This is the case for the flavoprotein ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena, an enzyme which interacts with ferredoxin in the photosynthetic pathway to receive the electrons required for NADP+ reduction. The reductase shows a concave cavity in its structure into which small proteins such as ferredoxin can fit. Flavodoxin, an FMN-containing protein that is synthesised in cyanobacteria under iron-deficient conditions, plays the same role as ferredoxin in its interaction with FNR in spite of its different structure, size and redox cofactor. There are a number of negatively charged amino acid residues on the surface of ferredoxin and flavodoxin that play a role in the electron transfer reaction with the reductase. Thus far, in only one case has charge replacement of one of the acidic residues produced an increase in the rate of electron transfer, whereas in several other cases a decrease in the rate is observed. In the most dramatic example, replacement of Glu at position 94 of Anabaena ferredoxin results in virtually the complete loss of ability to transfer electrons. Charge-reversal of positively charged amino acid residues in the reductase also produces strong effects on the rate of electron transfer. Several degrees of impairment have been observed, the most significant involving a positively charged Lys at position 75 which appears to be essential for the stability of the complex between the reductase and ferredoxin. The results presented in this paper provide a clear demonstration of the importance of electrostatic interactions on the stability of the transient complex formed during electron transfer by the proteins presently under study.     

Electrostatic and hydrophobic contributions to the folding mechanism of apocytochrome c driven by the interaction with lipid

In aqueous solution, while cytochrome c is a stably folded protein with a tightly packed structure at the secondary and tertiary levels, its heme-free precursor, apocytochrome c, shows all features of a structureless random coil. However, upon interaction with phospholipid vesicles or lysophospholipid micelles, apocytochrome c undergoes a conformational transition from its random coil in solution to an alpha-helical structure on association with lipid. The driving forces of this lipid-induced folding process of apocytochrome c were investigated for the interaction with various phospholipids and lysophospholipids. Binding of apocytochrome c to negatively charged phospholipid vesicles induced a partially folded state with approximately 85% of the alpha-helical structure of cytochrome c in solution. In contrast, in the presence of zwitterionic phospholipid vesicles, apocytochrome c remains a random coil, suggesting that negatively charged phospholipid headgroups play an important role in the mechanism of lipid-induced folding of apocytochrome c. However, negatively charged lysophospholipid micelles induce a higher content of alpha-helical structure than equivalent negatively charged diacylphospholipids in bilayers, reaching 100% of the alpha-helix content of cytochrome c in solution. Furthermore, micelles of lysolipids with the same zwitterionic headgroup of phospholipid bilayer vesicles induce approximately 60% of the alpha-helix content of cytochrome c in solution. On the basis of these results, we propose a mechanism for the folding of apocytochrome c induced by the interaction with lipid, which accounts for both electrostatic and hydrophobic contributions. Electrostatic lipid-protein interactions appear to direct the polypeptide to the micelle or vesicle surface and to induce an early partially folded state on the membrane surface. Hydrophobic interactions between nonpolar residues in the protein and the hydrophobic core of the lipid bilayer stabilize and extend the secondary structure upon membrane insertion.     

Flagellar motor-switch binding face of CheY and the biochemical basis of suppression by CheY mutants that compensate for motor-switch defects in Escherichia coli

CheY is a response regulator protein of Escherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis. The switch complex is composed of three proteins, FliG, FliM, and FliN. Recent biochemical data suggest a direct interaction of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY with limited allele specificity. The protein products of suppressor cheY alleles were purified and assayed for FliM binding. Six out of nine CheY mutants were defective in FliM binding. Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected if CheY suppressors were compensatory conformational suppressors. Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains. However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic. Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called "pseudotaxis." Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY. Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes.     

Isolation and in vitro characterization of CheZ suppressors for the Escherichia coli chemotactic response regulator mutant CheYN23D

The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein. We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sann, M.G., Swanson, R.V., Bourret, R.B., and Simon, M.I. (1995) Mol. Microbiol. 15, 1069-79). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.     

Design of fast enzymes by optimizing interaction potential in active site

The diffusional encounter between substrate and enzyme, and hence catalytic efficiency, can be enhanced by mutating charged residues on the surface of the enzyme. In this paper we present a simple method for screening such mutations. This is based on our earlier result that electrostatic enhancement of the enzyme-substrate binding rate constant can be accounted for just by the interaction potential within the active site. Assuming that catalytic and structural integrity is maintained, the catalytic efficiency can be optimized by surface charge mutations which lead to stronger interaction potential within the active site. Application of the screening method on superoxide dismutase shows that only charge mutations close to the active site will have practical effect on the catalytic efficiency. This rationalizes a large number of findings obtained in previous simulation and experimental studies.     

Mechanical and biological properties of two types of bioactive bone cements containing MgO-CaO-SiO2-P2O5-CaF2 glass and glass-ceramic powder

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 A radius centered in the enzyme monomer and separated from the protein surface by 1-14 A is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 A radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within +/- 10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase.     

Optimization of the electrostatic interactions between ionized groups and peptide dipoles in proteins

The three-dimensional optimization of the electrostatic interactions between the charged amino acid residues and the peptide partial charges was studied by Monte-Carlo simulations on a set of 127 nonhomologous protein structures with known atomic coordinates. It was shown that this type of interaction is very well optimized for all proteins in the data set, which suggests that they are a valuable driving force, at least for the native side-chain conformations. Similar to the optimization of the charge-charge interactions (Spassov VZ, Karshikoff AD, Ladenstein R, 1995, Protein Sci 4:1516-1527), the optimization effect was found more pronounced for enzymes than for proteins without enzymatic function. The asymmetry in the interactions of acidic and basic groups with the peptide dipoles was analyzed and a hypothesis was proposed that the properties of peptide dipoles are a factor contributing to the natural selection of the basic amino acids in the chemical composition of proteins.     

Collagen-induced arthritis in nonhuman primates: multiple epitopes of type II collagen can induce autoimmune-mediated arthritis in outbred cynomolgus monkeys

A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY approximately P) and the switch of the flagellar motor, located at its base. The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise. The docking site of CheY approximately P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17 fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products. The CheY-binding ability of each of the FliM mutant proteins was determined by chemical crosslinking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY approximately P to a much lesser extent than did wild-type FliM. CheY approximately P-binding of the other mutant proteins was similar to wild-type FliM. To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FliM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY approximately P. It also competed with full-length FliM on CheY approximately P. These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding.     

Functional changes in scavenger receptor binding conformation are induced by charge mutants spanning the entire collagen domain

Macrophage scavenger receptors are trimeric integral membrane proteins that bind a diverse array of negatively charged ligands. They have been shown to play a role in the pathogenesis of atherosclerosis and in host responses to microbial infections. Earlier mutational studies demonstrated that the distal segment of the collagen domain of the receptor was critically important for high affinity ligand binding activity. In this study, mutations spanning the entire collagen domain were generated and binding was assayed in transfected cells, as well as in assays employing a secreted, receptor fusion protein. Many of the distal, positively charged C-terminal residues in the type II collagen domain of the receptor, previously reported to be essential for binding at 37 degreesC, were found not to be critical for binding at 4 degreesC. Conversely, more proximally charged residues of the collagen receptor that have not been previously mutated were shown to have substantial effects on binding that were also temperature-dependent. These data suggest that scavenger receptor ligand recognition depends on more complex conformational interactions, involving charged residues throughout the entire collagen domain, than was previously recognized.     

Modeling of anti-nucleosome immunoglobulin Fv domains: analysis of electrostatic interactions

Three-dimensional structural models of six murine anti-(H2A-H2B) monoclonal autoantibody variable fragments were built by comparative molecular modeling using the COMPOSER software. Analysis of the antibody combining sites is based on the hypothesis that ionic and/or electrostatic interactions predominate in antigen antibody binding, as suggested by the cationic nature of histones and the amino acid sequences of the antibody hypervariable regions. The study of the electrostatic potentials of their combining site surfaces, computed with the MOLCAD software, and the comparison with the electrostatic potentials of 13 selected control mAbs show the lack of a unique electrostatic pattern. One group of three mAbs expresses a strong and large electronegative area, supporting the hypothesis that ionic interactions predominate in antigen recognition. The second group, containing the other three mAbs, exhibits an alternation of electropositive and electronegative areas. All, however, present a localized electronegative area in the vicinity of H-CDR1 and H-CDR2 loops that is generated by the presence of at least one acidic residue. The model suggesting that the binding activity may depend on charged residues at the same site is reminiscent of what was previously reported in anti-DNA mAbs. In addition, the alternation of electropositive areas and electronegative areas in second group mAbs is also frequently observed in certain anti-DNA mAbs. These data argue for the existence of relationships between these two autoantibody populations and suggest that they share a common immunogenic particle formed by anionic and cationic components, such as a nucleosome.