Modulation of substrate efflux in bacterial small multidrug resistance proteins by mutations at the dimer interface

Bacteria evade the effects of cytotoxic compounds through the efflux activity of membrane-bound transporters such as the small multidrug resistance (SMR) proteins. Consisting typically of ca. 110 residues with four transmembrane (TM) α-helices, crystallographic studies have shown that TM helix 1 (TM1) through TM helix 3 (TM3) of each monomer create a substrate binding "pocket" within the membrane bilayer, while a TM4-TM4 interaction accounts for the primary dimer formation. Previous work from our lab has characterized a highly conserved small-residue heptad motif in the Halobacterium salinarum transporter Hsmr as (90)GLXLIXXGV(98) that lies along the TM4-TM4 dimer interface of SMR proteins as required for function. Focusing on conserved positions 91, 93, 94, and 98, we substituted the naturally occurring Hsmr residue for Ala, Phe, Ile, Leu, Met, and Val at each position in the Hsmr TM4-TM4 interface. Large-residue replacements were studied for their ability to dimerize on SDS-polyacrylamide gels, to bind the cytotoxic compound ethidium bromide, and to confer resistance by efflux. Although the relative activity of mutants did not correlate with dimer strength for all mutants, all functional mutants lay within 10% of dimerization relative to the wild type (WT), suggesting that the optimal dimer strength at TM4 is required for proper efflux. Furthermore, nonfunctional substitutions at the center of the dimerization interface that do not alter dimer strength suggest a dynamic TM4-TM4 "pivot point" that responds to the efflux requirements of different substrates. This functionally critical region represents a potential target for inhibiting the ability of bacteria to evade the effects of cytotoxic compounds.     

Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations

Type I soluble inorganic pyrophosphatases (PPases) are well characterized both structurally and mechanistically. Earlier we measured the effects of active site substitutions on pH--rate profiles for the type I PPases from both Escherichia coli (E-PPase) and Saccharomyces cerevisae (Y-PPase). Here we extend these studies by measuring the effects of such substitutions on the more discrete steps of ligand binding to E-PPase, including (a) Mg(2+) and Mn(2+) binding in the absence of added ligand; (b) Mg(2+) binding in the presence of either P(i) or hydroxymethylbisphosphonate (HMBP), a competitive inhibitor of E-PPase; and (c) P(i) binding in the presence of Mn(2+). The active site of a type I PPase has well-defined subsites for the binding of four divalent metal ions (M1--M4) and two phosphates (P1, P2). Our results, considered in light of pertinent results from crystallographic studies on both E-PPase and Y-PPase and parallel functional studies on Y-PPase, allow us to conclude the following: (a) residues E20, D65, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural differences between the product and substrate complexes of E-PPase are concentrated in the lower half of the active site; (c) the M1 subsite is functionally isolated from the rest of the active site; and (d) the M4 subsite is an especially unconstrained part of the active site.     

Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.     

Disease-associated mutations at copper ligand histidine residues of superoxide dismutase 1 diminish the binding of copper and compromise dimer stability

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with (-)ONOO and H(2)O(2) in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind copper. We find that single mutations at copper ligands, including H46R, H48Q, and a quadruple mutant H46R/H48Q/H63G/H120G, also diminish the binding of radioactive copper. Further, using native polyacrylamide gel electrophoresis and a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry.     

Formyl-CoA transferase encloses the CoA binding site at the interface of an interlocked dimer

Formyl-CoA transferase catalyses transfer of CoA from formate to oxalate in the first step of oxalate degradation by Oxalobacter formigenes, a bacterium present in the intestinal flora which is implicated in oxalate catabolism in mammals. Formyl-CoA transferase is a member of a family of CoA-transferases for which no structural information is available. We now report the three-dimensional structure of O.formigenes formyl-CoA transferase, which reveals a novel fold and a very striking assembly of the homodimer. The subunit is composed of a large and a small domain where residues from both the N- and C-termini of the subunit are part of the large domain. The linkers between the domains give the subunit a circular shape with a hole in the middle. The enzyme monomers are tightly interacting and are interlocked. This fold requires drastic rearrangement of approximately 75 residues at the C-terminus for formation of the dimer. The structure of a complex of formyl-CoA transferase with CoA is also reported and sets the scene for a mechanistic understanding of enzymes of this family of CoA-transferases.     

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.     

Modulation of ligand binding by alternative splicing of the alphaPS2 integrin subunit

The Drosophila alphaPS2 integrin subunit is found in two isoforms. alphaPS2C contains 25 residues not found in alphaPS2m8, encoded by the alternative eighth exon. Previously, it was shown that cells expressing alphaPS2C spread more effectively than alphaPS2m8 cells on fragments of the ECM protein Tiggrin, and that alphaPS2C-containing integrins are relatively insensitive to depletion of Ca(2+). Using a ligand mimetic probe for Tiggrin affinity (TWOW-1), we show that the affinity of alphaPS2CbetaPS for this ligand is much higher than that of alphaPS2m8betaPS. However, the two isoforms become more similar in the presence of activating levels of Mn(2+). Modeling indicates that the exon 8-encoded residues replace the third beta strand of the third blade of the alpha subunit beta-propeller structure, and generate an exaggerated loop between this and the fourth strand. alphaPS2 subunits with the extra loop structure but with an m8-like third strand, or subunits with a C-like strand but an m8-like short loop, both fail to show alphaPS2C-like affinity for TWOW-1. Surprisingly, a single C > m8-like change at the third strand-loop transition point is sufficient to make alphaPS2C require Ca(2+) for function, despite the absence of any known cation binding site in this region. These data indicate that alternative splicing in integrin alpha subunit extracellular domains may affect ligand affinity via relatively subtle alterations in integrin conformation. These results may have relevance for vertebrate alpha6 and alpha7, which are alternatively spliced at the same site.     

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

Previous biochemical assays and a structural model of the protein have indicated that the dimer interface of the Hin recombinase is composed of two alpha-helices. To elucidate the structure and function of the helix, amino acids at the N-terminal end of the helix, where the two helices make their most extensive contact, were randomized, and inversion-incompetent mutants were selected. To investigate why the mutants lost their inversion activities, the DNA binding, hix pairing, invertasome formation, and DNA cleavage activities were assayed using in vivo and in vitro methodologies. The results indicated that the mutants could be divided into four classes based on their DNA binding activity. We propose that the alpha-helices might serve to place a DNA binding motif of Hin in the correct spatial relationship to the minor groove of the recombination site. All the mutants except those that failed to bind DNA were able to perform hix pairing and invertasome formation, suggesting that the dimer interface is not involved in either of these processes. The inversion-incompetent phenotype of the binders was caused by the inability of mutants to perform DNA cleavage. The mutants that showed less binding ability than the wild type nevertheless exhibited a wild-type level of hix pairing activity, because the hix pairing activity overcomes the defect in DNA binding. This phenotype of the mutants that are impaired in DNA binding suggests that the binding domains of Hin may mediate Hin-Hin interaction during hix pairing.     

Fusion deficiency induced by mutations at the dimer interface in the Newcastle disease virus hemagglutinin-neuraminidase is due to a temperature-dependent defect in receptor binding

The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37 degrees C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.     

Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase

Three Gln-80 residues belonging to different subunits of homohexameric Escherichia coli pyrophosphatase are separated by only one water molecule to which they are hydrogen bonded. Substitution of Glu for Gln-80 stabilizes quaternary structure of the enzyme but has only a small effect on enzyme activity. The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition. These data suggest that a new Mg2+ binding site is formed in the intersubunit contact region as a result of the substitution. Three-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues.     

Binding of substrate at the effector site of pyrophosphatase increases the rate of its hydrolysis at the active site

It is shown that in addition to the active site, each subunit of Escherichia coli inorganic pyrophosphatase (E-PPase) contains an extra binding site for the substrate magnesium pyrophosphate or its non-hydrolyzable analog magnesium methylenediphosphonate. The occupancy of the extra site stimulates the substrate conversion. Binding affinity of this site decreased or disappeared upon the conversion of E-PPase into a trimeric form or introduction of point mutations. However, when the slowly hydrolyzed substrate, lanthanum pyrophosphate (LaPP(i)), is used, the extra site was revealed in all enzyme forms of E-PPase and of Y-PPase (Saccharomyces cerevisiae PPase), resulting in about 100-fold activation of hydrolysis. A hypothesis on the localization of the extra site and the mechanism of its effect in E-PPase is presented.     

Alteration of the Fc gamma RIIa dimer interface affects receptor signaling but not ligand binding

The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcgammaRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcgammaRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcgammaRIIa may be an essential feature of the FcgammaRIIa signaling cascade.     

Functional clustering of mutations in the dimer interface of the nucleotide binding folds of the sulfonylurea receptor

ATP-sensitive K(+) (K(ATP)) channels modulate their activity as a function of inhibitory ATP and stimulatory Mg-nucleotides. They are constituted by two proteins: a pore-forming K(+) channel subunit (Kir6.1, Kir6.2) and a regulatory sulfonylurea receptor (SUR) subunit, an ATP-binding cassette (ABC) transporter that confers MgADP stimulation to the channel. Channel regulation by MgADP is dependent on nucleotide interaction with the cytoplasmic nucleotide binding folds (NBF1 and NBF2) of the SUR subunit. Crystal structures of bacterial ABC proteins indicate that NBFs form as dimers, suggesting that NBF1-NBF2 heterodimers may form in SUR and other eukaryotic ABC proteins. We have modeled SUR1 NBF1 and NBF2 as a heterodimer, and tested the validity of the predicted dimer interface by systematic mutagenesis. Engineered cysteine mutations in this region have significant effects, both positive and negative, on MgADP stimulation of K(ATP) channels in excised patches and on macroscopic channel activity in intact cells. Additionally, the mutations cluster in the model structure according to their functional effect, such that patterns of alteration emerge. Of note, three gain-of-function mutations, leading to MgADP hyperstimulation of the channel, are located in the D-loop region at the center of the predicted dimer interface. Overall, the data support the idea that SUR1 NBFs assemble as heterodimers and that this interaction is functionally critical.     

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.     

Effectory site in Escherichia coli inorganic pyrophosphatase is revealed upon mutation at the intertrimeric interface

Escherichia coli inorganic pyrophosphatase (E-PPase) is a homohexamer formed from two trimers related by a two-fold axis. The residue Asp26 participates in intertrimeric contacts. Kinetics of MgPPi hydrolysis by a mutant Asp26Ala E-PPase is found to not obey Michaelis-Menten equation but can be described within the scheme of activation of hydrolysis by a free PPi binding at an effectory subsite. Existence of such a subsite is confirmed by the finding that the free form of methylenediphosphonate activates MgPPi hydrolysis though its magnesium complex is a competitive inhibitor. The Asp26Ala variant is the first example of hexameric E-PPase demonstrated to have an activatory subsite.     

Evolutionary trace analysis at the ligand binding site of laccase

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.     

Shift of binding site at the interface between actin and myosin

The molar ratio dependent change in the binding manner between actin and the lysine-rich sequence at the junction between 50K and 20K domains of subfragment 1 was studied by both protease digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The tryptic cleavage site at the function between 50K and 20K was found to be located between the third and fourth lysine residues in the lysine-rich sequence -KKGGKKK-. This site was not protected by actin when the molar ratio of actin to subfragment 1 was 1:1 but was protected at 2:1 and 3:1. The V8 protease cleavage site of chicken subfragment 1 and the elastase cleavage site of rabbit subfragment 1 were found to be located four residues away from the N-terminus of the lysine-rich sequence. Unlike the tryptic cleavage site, this site was protected by actin more when the molar ratio of actin to subfragment 1 was 1:1 than when it was 2:1 and 3:1. To understand the reason for the opposite effect of the molar ratio observed at the middle of and at four residues away from the lysine-rich sequence, actual cross-linked residue(s) was (were) determined by subjecting cross-linked product to a protein sequencer. It was found that the cross-linked sites were mainly at the first and second lysine residues of the lysine-rich sequence when the molar ratio of actin to subfragment 1 was 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)