Multistate allostery in response regulators: phosphorylation and mutagenesis activate RegA via alternate modes

Response regulators (RRs) belong to two-component signaling pathways, widely prevalent in bacteria and lower eukaryotes, for sensing and mediating responses to diverse environmental stress stimuli. RRs are modular proteins, and in most instances, a receiver domain is found connected to diverse effector domain(s). All receiver domains contain a conserved aspartate, which is the site of phosphorylation by an associated histidine kinase. RRs function as phosphorylatable signaling switches whereby histidine-kinase-mediated phosphorylation of RRs alters its output function. It is largely unknown how phosphorylation of the receiver domain triggers activation of distally positioned effector domain(s). Although crystal structures have highlighted differences in conformations from comparisons of snapshots of the unphosphorylated and phosphorylated receiver domains, how this is translated into altered activity of a distal effector domain has remained a mystery. While allosteric relays have been identified within receiver domains by NMR and X-ray crystallography, phosphorylated states of larger multidomain RRs have not yet been characterized. In this study, we have used amide hydrogen/deuterium exchange mass spectrometry to probe the conformational dynamics of a multidomain RR, RegA from Dictyostelium discoideum, by comparisons of the unphosphorylated and phosphorylated states and an activating mutant. Our results reveal allosteric coupling between the site of phosphorylation and the activating mutation. Interestingly, however, the conformations of the effector domains in both instances are distinct. Hydrogen/deuterium exchange mass spectrometry indicates that the 'inactive' and 'active' conformations exist as ensembles of multiple conformations. This is consistent with the 'conformational selection' model for describing phosphorylation-dependent regulation of multidomain RRs.     

Biochemical evidence for multiple dimeric states of the Sinorhizobium meliloti DctD receiver domain

X-ray crystal structures suggest very different dimeric states for the inactive and active forms of the two-component receiver domain of Sinorhizobium meliloti DctD, a sigma(54)-dependent AAA+ ATPase. Moreover, the receiver domain in crystals grown from unphosphorylated protein is refractory to phosphorylation whereas solution protein is fully phosphorylatable, and equilibrium analytical ultracentrifugation data are consistent with solution dimers for both phosphorylated and unphosphorylated forms of the protein. Here we report biochemical data consistent with the presence of multiple dimeric conformations in the inactive and active states, and evidence for significant change in the dimeric state upon activation by phosphorylation or binding of Mg(2+) and BeF(3)(-).     

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.     

The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp‐encoded regulator SypA in Vibrio fischeri

Bacteria utilize multiple regulatory systems to modulate gene expression in response to environmental changes, including two‐component signalling systems and partner‐switching networks. We recently identified a novel regulatory protein, SypE, that combines features of both signalling systems. SypE contains a central response regulator receiver domain flanked by putative kinase and phosphatase effector domains with similarity to partner‐switching proteins. SypE was previously shown to exert dual control over biofilm formation through the opposing activities of its terminal effector domains. Here, we demonstrate that SypE controls biofilms in Vibrio fischeri by regulating the activity of SypA, a STAS (sulphate transporter and anti‐sigma antagonist) domain protein. Using biochemical and genetic approaches, we determined that SypE both phosphorylates and dephosphorylates SypA, and that phosphorylation inhibits SypA's activity. Furthermore, we found that biofilm formation and symbiotic colonization required active, unphosphorylated SypA, and thus SypA phosphorylation corresponded with a loss of biofilms and impaired host colonization. Finally, expression of a non‐phosphorylatable mutant of SypA suppressed both the biofilm and symbiosis defects of a constitutively inhibitory SypE mutant strain. This study demonstrates that regulation of SypA activity by SypE is a critical mechanism by which V. fischeri controls biofilm development and symbiotic colonization.     

Crystal structure of receiver domain of putative NarL family response regulator spr1814 from Streptococcus pneumoniae in the absence and presence of the phosphoryl analog beryllofluoride

Spr1814 of Streptococcus pneumoniae is a putative response regulator (RR) that has four-helix helix-turn-helix DNA-binding domain and belongs to the NarL family. The prototypical RR contains two domains, an N-terminal receiver domain linked to a variable effector domain. The receiver domain functions as a phosphorylation-activated switch and contains the typical doubly wound five-stranded α/β fold. Here, we report the crystal structure of the receiver domain of spr1814 (spr1814(R)) determined in the absence and presence of beryllofluoride as a phosphoryl analog. Based on the overall structure, spr1814(R) was shown to contain the typical fold similar with other structures of the receiver domain; however, an additional linker region connecting the receiver and DNA-binding domain was inserted into the dimer interface of spr1814(R), resulting in the formation of unique dimer interface. Upon phosphorylation, the conformational change of the linker region was observed and this suggests that domain rearrangement between the receiver domain and effector domain could occur in full-length spr1814.     

Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer,Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions

The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp⁵⁰ residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called “phosphorylation pocket” is not conserved, with its space being occupied by an Arg⁵² from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp⁵⁰ with the highly conserved Arg⁵² and Thr⁹ in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.     

Molecular Dynamic Simulations of the N-Terminal Receiver Domain of NtrC Reveal Intrinsic Conformational Flexibility in the Inactive State

Abstract

The N-terminal receiver domain of NtrC is the molecular switch in the two-component signal transduction. It is the first protein where structures of both the active (phosphyroylated) and inactive (unphosphyroylated) states are determined experimentally. Phosphorylation of the NtrC at the active site induces large structural change. NMR experiments suggested that the wild type unphosphorylated NtrC adopts both the active and the inactive conformations and the phosphorylation stabilizes the active conformations. We applied free (unconstrained) molecular dynamic (MD) simulation to examine the intrinsic flexibilities and stabilities of the NtrC receiver domain in both the active and inactive conformations. Molecular dynamic simulations showed that the inactive state of NtrC receiver domain is more flexible than the active state. There were large movements in helix 4 and loop β3-α3 which coincide with major structural differences between the inactive and active states. We observed large root-mean-square deviations from the initial starting structure and the large root-mean-square fluctuations during MD simulation for the inactive state. We then investigated the activation pathway with Targeted MD simulation. We show that the intrinsic flexibility in the loop β3-α3 plays an important role in triggering the conformational change. Phosphorylation at the active site may serve to stabilize the conformational change. These results together suggest that the unphosphorylated NtrC receiver domain could be involved in a conformational equilibrium between two different states.     

The structure of a full-length response regulator from Mycobacterium tuberculosis in a stabilized three-dimensional domain-swapped, activated state

The full-length, two-domain response regulator RegX3 from Mycobacterium tuberculosis is a dimer stabilized by three-dimensional domain swapping. Dimerization is known to occur in the OmpR/PhoB subfamily of response regulators upon activation but has previously only been structurally characterized for isolated receiver domains. The RegX3 dimer has a bipartite intermolecular interface, which buries 2357 A(2) per monomer. The two parts of the interface are between the two receiver domains (dimerization interface) and between a composite receiver domain and the effector domain of the second molecule (interdomain interface). The structure provides support for the importance of threonine and tyrosine residues in the signal transduction mechanism. These residues occur in an active-like conformation stabilized by lanthanum ions. In solution, RegX3 exists as both a monomer and a dimer in a concentration-dependent equilibrium. The dimer in solution differs from the active form observed in the crystal, resembling instead the model of the inactive full-length response regulator PhoB.     

Dimer-induced signal propagation in Spo0A

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

Cytochrome c₅₅₅ from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c₅₅₅) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 3₁₀‐α‐3₁₀ helix containing the heme‐ligating Met61. Herein, we show that AA cyt c₅₅₅ forms dimers by swapping the region containing the extra 3₁₀‐α‐3₁₀ helix and C‐terminal α‐helix. The asymmetric unit of the crystal of dimeric AA cyt c₅₅₅ contained two dimer structures, where the structure of the hinge region (Val53–Lys57) was different among all four protomers. Dimeric AA cyt c₅₅₅ dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c₅₅₅ were maintained at pH 2.2–11.0. CN^‐ and CO bound to dimeric AA cyt c₅₅₅ in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN^‐ and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2–11.0), respectively, in a wide range of temperature (25–85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c₅₅₅ changes upon dimerization by domain swapping.