Changes In Conformational Order Research Articles

The Dynamical Properties of Three Different Variants of the Orange Carotenoid Protein: A Quasielastic Neutron Scattering Study

Besides a well-adapted structure, proteins often require a specific dynamical flexibility to undergo conformational changes in order to carry out their function. The latter dynamics can be directly measured by quasielastic neutron scattering as demonstrated here for three variants of the orange carotenoid protein (OCP), which plays a pivotal role in the protection of the cyanobacterial photosynthetic apparatus against photodamage. We investigate the dynamics of the structurally compact, dark-adapted wild type of OCP (OCPwt) in comparison with that of two mutant forms. The latter two mutants differ preferentially in their structures. The orange mutant OCP-W288A is assumed to have a compact structure and to preferentially bind the pigment echinenone, while the pink mutant OCP-W288A appears to represent the more elongated structure of the red active state of OCP binding the carotenoid canthaxanthin, respectively. The study reveals three major findings: (a) the dynamics of the red active state of OCP is significantly enhanced due to a larger number of protein residues being exposed to the solvent at the surface of the protein; (b) the dynamics of all OCP forms appear to be suppressed upon the freezing of the solvent, which is most likely due to an ice-induced aggregation of the proteins; and (c) the wild type and the compact mutant exhibit different dynamics attributed to a missing H-bond between the pigment and protein, resulting a destabilization of the surrounding protein.

Structure of HIV-1 gp41 with its membrane anchors targeted by neutralizing antibodies.

The HIV-1 gp120/gp41 trimer undergoes a series of conformational changes in order to catalyze gp41-induced fusion of viral and cellular membranes. Here, we present the crystal structure of gp41 locked in a fusion intermediate state by an MPER-specific neutralizing antibody. The structure illustrates the conformational plasticity of the six membrane anchors arranged asymmetrically with the fusion peptides and the transmembrane regions pointing into different directions. Hinge regions located adjacent to the fusion peptide and the transmembrane region facilitate the conformational flexibility that allows high-affinity binding of broadly neutralizing anti-MPER antibodies. Molecular dynamics simulation of the MPER Ab-stabilized gp41 conformation reveals a possible transition pathway into the final post-fusion conformation with the central fusion peptides forming a hydrophobic core with flanking transmembrane regions. This suggests that MPER-specific broadly neutralizing antibodies can block final steps of refolding of the fusion peptide and the transmembrane region, which is required for completing membrane fusion.

Conformational Landscape of Cytochrome P450 Reductase Interactions.

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein–protein docking to investigate the conformational changes necessary for the formation of the CPR–CYP complex. While some structural features of the CPR and the CPR–CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR–CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR–CYP interactions and underlying conformational changes, improving our understanding of this complex machinery relevant for drug metabolism.

Adsorption of n-alkanes in ZIF-8: Influence of crystal size and framework dynamics

Due to its exceptional chemical and thermal stability, ZIF-8 is one of the most promising representatives of nanoporous metal-organic frameworks. In this work, we investigate adsorption properties of this material both experimentally and theoretically. The experiments were carried out on 8 preparations differing in morphology of the crystals. Adsorption was studied in isothermal approach exploiting standard adsorbates, such as N 2 or CO 2 , as well as in isobaric regime with C5–C9 linear alkanes. The latter were performed with the novel quasi-equilibrated temperature-programmed desorption and adsorption (QE-TPDA) technique showing that a complexity of n-alkanes adsorption mechanism in ZIF-8 depends on the nature of adsorbate. Unexpectedly, for adsorption of C7–C9 n-alkanes a two-step process was found. QE-TPDA yielded high quality adsorption isobars which were successfully reproduced by Grand-Canonical Monte Carlo molecular simulations. The calculations showed that the specific adsorption behaviour of ZIF-8 is due to the fact that its structure undergoes conformational changes in order to adapt to the guest molecules. QE-TPDA measurements with n-nonane were performed at conditions close to saturation of the adsorbate. This allowed to observe surface-related adsorption on the ZIF-8 crystals, which was correlated with their size. • Combined theoretical and experimental studies on adsorption of C5–C9 n-alkanes in ZIF-8. • Investigations on the influence of crystal size on sorption properties. • Two-step adsorption of specific n-alkanes influenced by framework flexibility. • Packing of n-alkanes similar as for LTA-type zeolites. • Hyperbolic trend between surface adsorption amount and the size of the crystals.

Structural Basis of Protein Kinase C Tumor‐Promoting Response And Membrane Recruitment

Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. The hallmark of their activation is translocation to lipid membranes. Dysregulation of PKC signaling has been implicated in cancer progression, cardiac disease, and Alzheimer’s disease. One of the major challenges in the field is to understand the structural basis of PKC activation and regulation ‐‐ at the level of atomic resolution.Here, we applied NMR and FRET spectroscopy to gain insight into the structure and protein‐membrane interactions of the regulatory domain of PKC that consist of three peripheral membrane modules: the tandem conserved homology‐1 (C1) and conserved homology‐2 (C2) domains. The C1 domains undergo membrane insertion upon binding their natural agonist, diacylglycerol, as well as tumor‐promoting phorbol esters and other small‐molecule agonists. C2 associates with anionic membranes upon binding Ca2+. Simultaneous association of all domains with membranes ensures the coincidence of four signals: Ca2+ ions, diacylglycerol, phosphatidylserine, and phosphatidylinositol‐4,5‐bisphosphate.Here, we report the first structure of the C1 domain complexed to a potent tumor‐promoting agent, phorbol 12,13‐dibutyrate (PDBu), in the membrane‐mimicking environment. The structure of the complex shows that C1 has to undergo a conformational change in order to capture the membrane‐embedded ligand. The structure also revealed the role of individual amino acids in the interactions with PDBu and membrane‐mimicking environment. To probe how different agonists modulate the interaction of C1 with membranes, we conducted NMR paramagnetic relaxation enhancement (PRE) experiments with bicelles doped with paramagnetic lipid. The PRE patterns revealed that the C1 domains adopt tilted rather than vertical orientation in the membrane. Our data on four different agonists indicate that C1 domains dictate the general orientation of the agonist complexes in the membrane, but the agonists determine the depth of insertion. This provides a plausible explanation for the differences in the potency of these ligands to achieve the acute and chronic activation response of PKC.To gain insight into the mechanism of coincidence detection, we developed protein‐to‐membrane FRET assay that we used to characterize the interplay of the C1 and C2 domains in the membrane association step. The FRET experiments revealed a clear picture of how tumor‐promoting agents can elicit constitutive activation of PKC through simultaneous recruitment of C1 domains to membranes and sensitization of the C2 domain to Ca2+.Support or Funding InformationNIH R01 GM108998; NSF CHE‐1905116; Welch Foundation A‐1784Agonist‐guided membrane interaction of C1B domain of PKCδ studied by paramagnetic NMR. (A) Schematic representation of the approach. (B) Chemical representations of the different agonists used in the study.Figure 1

An Investigation of the Influenza Hemagglutinin Membrane Fusion Process using Microsecond-Level MD Simulations

Influenza virus hemagglutinin (HA) is a homotrimeric envelope protein that undergoes pH-triggered conformational changes in order to facilitate the fusion of the viral envelope with the endosomal membrane of the infected host cell. Previous experimental studies have outlined a mechanism for the conformational transitions that lead to membrane fusion. At low pH, the HA1 chains move away from the HA2 chains, which then undergo a series of conformational changes to expose the N-terminal fusion peptide. The HA2 chains then bend at a hinge region to form a hairpin shaped structure, thus bringing the viral envelope into contact with the endosomal membrane. While this model is well established, the conformational changes have never been observed in all-atom equilibrium MD simulations. Using a combination of equilibrium MD simulations and non-equilibrium SMD simulations, we have characterized the conformational dynamics of the membrane fusion process. Six models were simulated for 2.4 microseconds each. By modifying the protonation state of a conserved histidine in the HA2 hinge region, we have successfully simulated the pH-mediated conformational changes that occur when HA1 moves away from HA2. Our atomistic MD simulations confirmed that the HA1 chain associated with an HA2 chain moves away only when the histidine is protonated. Using non-equilibrium pulling simulations, we have also modeled the complex conformational and secondary-structural transitions that lead to the uncoiling of HA2 and the exposure of the fusion peptide. In addition to the conformational changes that occur within hemagglutinin, we have independently modeled the fusion of the viral envelope with the host cell endosomal membrane. For the very first time, we have studied the conformational dynamics of the entire HA-membrane complex using microsecond-level atomistic MD simulations and non-equilibrium simulations.

Reactive centre loop dynamics and serpin specificity

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

Investigating the Conformational Dynamics of the Membrane Enzyme LspA

Lipoprotein signal peptidase (LspA) is a key enzyme in the lipoprotein processing pathway and is responsible for cleaving the signal sequence of prolipoproteins. LspA is a membrane embedded enzyme that is hypothesized to undergo conformational changes in order for the transmembrane alpha-helical substrate to enter the active site. As both the enzyme and substrate reside in the bacterial inner membrane, enzyme-lipid interactions likely play a role in modulating these conformational changes. LspA conformation changes upon substrate binding and in different membrane mimics are investigated using electron paramagnetic resonance (EPR). Key residues involved in protein - protein interactions and protein - lipid interactions identified using evolutionary couplings are explored using mutagenesis and differing lipid composition. As LspA has been identified as a good candidate for an antibacterial drug target, these studies will also aid in future structure-guided drug design.

Structural Immunology of Complement Receptors 3 and 4.

Complement receptors (CR) 3 and 4 belong to the family of beta-2 (CD18) integrins. CR3 and CR4 are often co-expressed in the myeloid subsets of leukocytes, but they are also found in NK cells and activated T and B lymphocytes. The heterodimeric ectodomain undergoes considerable conformational change in order to switch the receptor from a structurally bent, ligand-binding in-active state into an extended, ligand-binding active state. CR3 binds the C3d fragment of C3 in a way permitting CR2 also to bind concomitantly. This enables a hand-over of complement-opsonized antigens from the cell surface of CR3-expressing macrophages to the CR2-expressing B lymphocytes, in consequence acting as an antigen presentation mechanism. As a more enigmatic part of their functions, both CR3 and CR4 bind several structurally unrelated proteins, engineered peptides, and glycosaminoglycans. No consensus motif in the proteinaceous ligands has been established. Yet, the experimental evidence clearly suggest that the ligands are primarily, if not entirely, recognized by a single site within the receptors, namely the metal-ion dependent adhesion site (MIDAS). Comparison of some recent identified ligands points to CR3 as inclined to bind positively charged species, while CR4, by contrast, binds strongly negative-charged species, in both cases with the critical involvement of deprotonated, acidic groups as ligands for the Mg2+ ion in the MIDAS. These properties place CR3 and CR4 firmly within the realm of modern molecular medicine in several ways. The expression of CR3 and CR4 in NK cells was recently demonstrated to enable complement-dependent cell cytotoxicity toward antibody-coated cancer cells as part of biological therapy, constituting a significant part of the efficacy of such treatment. With the flexible principles of ligand recognition, it is also possible to propose a response of CR3 and CR4 to existing medicines thereby opening a possibility of drug repurposing to influence the function of these receptors. Here, from advances in the structural and cellular immunology of CR3 and CR4, we review insights on their biochemistry and functions in the immune system.

Methotrexate and Cytarabine-Loaded Nanocarriers for Multidrug Cancer Therapy. Spectroscopic Study.

Determining the properties of nanoparticles obtained by novel methods and defining the scope of their application as drug carriers has important practical significance. This article presents the pioneering studies concerning high degree incorporation of cytarabine (AraC) and methotrexate (MTX) into liposome vesicles. The main focus of this study were cytarabine-methotrexate-dipalmitoylphosphatidylcholine (DPPC) interactions observed in the gel and fluid phases of DPPC bilayers. The proposed new method of use the Transmittance2919/2850 ratio presented in our research is sensitive to subtle changes in conformational order resulting from rotations, kinks and bends of the lipid chains. The transition temperatures characterized by Fourier Transform Infrared Spectroscopy (FT-IR) were consistent with the results obtained by Differential Scanning Calorimetry (DSC). Transmission Electron Microscopy (TEM) was used in order to determine the size and shape of the liposomes obtained. The mutual interactions occurring between the drugs studied and the phospholipids were analyzed using the Nuclear Magnetic Resonance (NMR).

Smoothing a rugged protein folding landscape by sequence-based redesign.

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.

Review: Mechanochemistry of the kinesin-1 ATPase.

ABSTRACTKinesins are P‐loop NTPases that can do mechanical work. Like small G‐proteins, to which they are related, kinesins execute a program of active site conformational changes that cleaves the terminal phosphate from an NTP substrate. But unlike small G‐proteins, kinesins can amplify and harness these conformational changes in order to exert force. In this short review I summarize current ideas about how the kinesin active site works and outline how the active site chemistry is coupled to the larger‐scale structural cycle of the kinesin motor domain. Focusing largely on kinesin‐1, the best‐studied kinesin, I discuss how the active site switch machinery of kinesin cycles between three distinct states, how docking of the neck linker stabilizes two of these states, and how tension‐sensitive and position‐sensitive neck linker docking may modulate both the hydrolysis step of ATP turnover and the trapping of product ADP in the active site. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 476–482, 2016.

Crystal structure of recombinant tyrosinase-binding protein MtaL at 1.35 Å resolution.

Mushroom tyrosinase-associated lectin-like protein (MtaL) binds to mature Agaricus bisporus tyrosinase in vivo, but the exact physiological function of MtaL is unknown. In this study, the crystal structure of recombinant MtaL is reported at 1.35 Å resolution. Comparison of its structure with that of the truncated and cleaved MtaL present in the complex with tyrosinase directly isolated from mushroom shows that the general β-trefoil fold is conserved. However, differences are detected in the loop regions, particularly in the β2-β3 loop, which is intact and not cleaved in the recombinant MtaL. Furthermore, the N-terminal tail is rotated inwards, covering the tyrosinase-binding interface. Thus, MtaL must undergo conformational changes in order to bind mature mushroom tyrosinase. Very interestingly, the β-trefoil fold has been identified to be essential for carbohydrate interaction in other lectin-like proteins. Comparison of the structures of MtaL and a ricin-B-like lectin with a bound disaccharide shows that MtaL may have a similar carbohydrate-binding site that might be involved in glycoreceptor activity.

Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two α-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 Å resolution) shows typical features of group-II truncated hemoglobins, namely the two-on-two α-helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment.

Chemistry in motion: tiny synthetic motors.

CONSPECTUS: Diffusion is the principal transport mechanism that controls the motion of solute molecules and other species in solution; however, the random walk process that underlies diffusion is slow and often nonspecific. Although diffusion is an essential mechanism for transport in the biological realm, biological systems have devised more efficient transport mechanisms using molecular motors. Most biological motors utilize some form of chemical energy derived from their surroundings to induce conformational changes in order to carry out specific functions. These small molecular motors operate in the presence of strong thermal fluctuations and in the regime of low Reynolds numbers, where viscous forces dominate inertial forces. Thus, their dynamical behavior is fundamentally different from that of macroscopic motors, and different mechanisms are responsible for the production of useful mechanical motion. There is no reason why our interest should be confined to the small motors that occur naturally in biological systems. Recently, micron and nanoscale motors that use chemical energy to produce directed motion by a number of different mechanisms have been made in the laboratory. These small synthetic motors also experience strong thermal fluctuations and operate in regimes where viscous forces dominate. Potentially, these motors could be directed to perform different transport tasks, analogous to those of biological motors, for both in vivo and in vitro applications. Although some synthetic motors execute conformational changes to effect motion, the majority do not, and, instead, they use other mechanisms to convert chemical energy into directed motion. In this Account, we describe how synthetic motors that operate by self-diffusiophoresis make use of a self-generated concentration gradient to drive motor motion. A description of propulsion by self-diffusiophoresis is presented for Janus particle motors comprising catalytic and noncatalytic faces. The properties of the dynamics of chemically powered motors are illustrated by presenting the results of particle-based simulations of sphere-dimer motors constructed from linked catalytic and noncatalytic spheres. The geometries of both Janus and sphere-dimer motors with asymmetric catalytic activity support the formation of concentration gradients around the motors. Because directed motion can occur only when the system is not in equilibrium, the nature of the environment and the role it plays in motor dynamics are described. Rotational Brownian motion also acts to limit directed motion, and it has especially strong effects for very small motors. We address the following question: how small can motors be and still exhibit effects due to propulsion, even if only to enhance diffusion? Synthetic motors have the potential to transform the manner in which chemical dynamical processes are carried out for a wide range of applications.

Conformational changes in DNA‐binding proteins: Relationships with precomplex features and contributions to specificity and stability

Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA-binding proteins by superimposing DNA-bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA-binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner.

SHP Family Protein Tyrosine Phosphatases Adopt Canonical Active-Site Conformations in the Apo and Phosphate-Bound States

Protein tyrosine phosphatase (PTP) catalytic domains undergo a series of conformational changes in order to mediate dephosphorylation of their tyrosine phosphorylated substrates. An important conformational change occurs in the Tryptophan-Proline-Aspartic acid (WPD) loop, which contains the conserved catalytic aspartate. Upon substrate binding, the WPD loop transitions from the 'open' to the 'closed' state, thus allowing optimal positioning of the catalytic aspartate for substrate dephosphorylation. The dynamics of WPD loop conformational changes have previously been studied for PTP1B, HePTP, and the bacterial phosphatase YopH, but have not yet been comprehensively studied for the nonreceptor tyrosine phosphatase SHP-1 (PTPN6). To structurally describe the changes in WPD loop conformation in SHP-1, we have determined the 1.4 Å crystal structure of the catalytic domain of SHP-1 in the Apo state and the 1.8 Å crystal structure of the SHP-1 catalytic domain in complex with a phosphate ion. We provide structural analysis for the WPD loop closed state of SHP phosphatases and the conformational changes that occur upon WPD loop closure.

Variability in Fusogenic Activity of Human Metapneumovirus Fusion Protein from Different Strains

First identified in 2001, the paramyxovirus human metapneumovirus (HMPV) is a novel pathogen that causes viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide and is the second most common cause of pediatric lower respiratory illness, following the closely related RSV. Despite its clinical significance, little is known about its entry pathway, complicating the search for antivirals. Of the three surface glycoproteins expressed on the viral membrane, the attachment protein, a small hydrophobic protein, and the fusion protein F, only the F protein is required for membrane fusion and infectivity. The F protein undergoes a dramatic conformational change in order to bring the viral and target cell membranes together. This conformation change in HMPV strain CAN97–83 has been shown to be triggered by low pH. However, low pH triggering varies between F proteins of different strains. In this study we characterized fusogenic activity of strains representative of all four clades using two independent fusion assays, syncytia formation and luciferase reporter gene assay. Our results suggest that both the overall fusion level and the amount of stimulation by low pH vary between F proteins of different strains. Comparison of F protein sequence will be used to identify potential critical residues. This research was supported by NIH grant R01AI051517.

The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH

Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression. We present here the 2.2 Å X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA. We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.

The Crystal Structure of AphB, a Virulence Gene Activator from Vibrio Cholerae, Reveals Residues that Influence its Response to Oxygen and pH

For Vibrio cholerae O1 to cause disease, it must express two critical virulence factors: the toxin-coregulated pilus and cholera toxin. Expression of these virulence factors is controlled by a transcriptional cascade initiated at the tcpPH promoter by the regulatory proteins AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate expression of the tcpPH promoter. We present here the 2.2 A X-ray crystal structure of full-length AphB. As reported for several other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order for DNA binding to occur. Although a natural ligand for AphB has not yet been identified, we have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate transcription of tcpPH at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that the activity of AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB, suggesting a mechanism by which ligand binding leads to activation of the AphB-tcpPH promoter complex.