Crystal Structures of the Carboxyl cGMP Binding Domain of the Plasmodium falciparum cGMP-dependent Protein Kinase Reveal a Novel Capping Triad Crucial for Merozoite Egress

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation.     

ST1710–DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators

ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 Å resolutions, respectively. The ST1710–DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710–DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism.     

Crystal structure and function of the zinc uptake regulator FurB from Mycobacterium tuberculosis

Members of the ferric/zinc uptake regulator (Fur/Zur) family are the central metal-dependent regulator proteins in many Gram-negative and -positive bacteria. They are responsible for the control of a wide variety of basic physiological processes and the expression of important virulence factors in human pathogens. Therefore, Fur has gathered significant interest as a potential target for novel antibiotics. Here we report the crystal structure of FurB from Mycobacterium tuberculosis at a resolution of 2.7A, and we present biochemical and spectroscopic data that allow us to propose the functional role of this protein. Although the overall fold of FurB with an N-terminal DNA binding domain and a C-terminal dimerization domain is conserved among the Zur/Fur family, large differences in the spatial arrangement of the two domains with respect to each other can be observed. The biochemical and spectroscopic analysis presented here reveals that M. tuberculosis FurB is Zn(II)-dependent and is likely to control genes involved in the bacterial zinc uptake. The combination of the structural, spectroscopic, and biochemical results enables us to determine the structural basis for functional differences in this important family of bacterial regulators.     

Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State

Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.     

Crystal Structure of Peroxide Stress Regulator from Streptococcus pyogenes Provides Functional Insights into the Mechanism of Oxidative Stress Sensing

Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.     

Crystal Structure of the Pneumococcal Vancomycin-Resistance Response Regulator DNA-Binding Domain

Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 _Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 _Å, b = 52.97 _Å, and c = 60.61 _Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.     

SpoIIIE Protein Achieves Directional DNA Translocation through Allosteric Regulation of ATPase Activity by an Accessory Domain

Bacterial chromosome segregation utilizes highly conserved directional translocases of the SpoIIIE/FtsK family. These proteins employ an accessory DNA-binding domain (γ) to dictate directionality of DNA transport. It remains unclear how the interaction of γ with specific recognition sequences coordinates directional DNA translocation. We demonstrate that the γ domain of SpoIIIE inhibits ATPase activity of the motor domain in the absence of DNA but stimulates ATPase activity through sequence-specific DNA recognition. Furthermore, we observe that communication between γ subunits is necessary for both regulatory roles. Consistent with these findings, the γ domain is necessary for robust DNA transport along the length of the chromosome in vivo. Together, our data reveal that directional activation involves allosteric regulation of ATP turnover through coordinated action of γ domains. Thus, we propose a coordinated stimulation model in which γ-γ communication is required to translate DNA sequence information from each γ to its respective motor domain.     

Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography

Ferric uptake regulator (Fur) is a global bacterial regulator that uses iron as a cofactor to bind to specific DNA sequences. Escherichia coli Fur is usually isolated as a homodimer with two metal sites per subunit. Metal binding to the iron site induces protein activation; however the exact role of the structural zinc site is still unknown. Structural studies of three different forms of the Escherichia coli Fur protein (nonactivated dimer, monomer, and truncated Fur-(1-82)) were performed. Dimerization of the oxidized monomer was followed by NMR in the presence of a reductant (dithiothreitol) and Zn(II). Reduction of the disulfide bridges causes only local structure variations, whereas zinc addition to reduced Fur induces protein dimerization. This demonstrates for the first time the essential role of zinc in the stabilization of the quaternary structure. The secondary structures of the mono- and dimeric forms are almost conserved in the N-terminal DNA-binding domain, except for the first helix, which is not present in the nonactivated dimer. In contrast, the C-terminal dimerization domain is well structured in the dimer but appears flexible in the monomer. This is also confirmed by heteronuclear Overhauser effect data. The crystal structure at 1.8A resolution of a truncated protein (Fur-(1-82)) is described and found to be identical to the N-terminal domain in the monomeric and in the metal-activated state. Altogether, these data allow us to propose an activation mechanism for E. coli Fur involving the folding/unfolding of the N-terminal helix.     

The cAMP Receptor-Like Protein CLP Is a Novel c-di-GMP Receptor Linking Cell-Cell Signaling to Virulence Gene Expression in Xanthomonas campestris

Cyclic-di-GMP [bis-(3′-5′)-cyclic diguanosine monophosphate] controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris, expression of a subset of virulence genes is regulated by c-di-GMP and also by the CAP (catabolite activation protein)-like protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene expression. XcCLP bound target promoter DNA with submicromolar affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with micromolar affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-di-GMP. Together, these findings reveal the structural mechanism behind a novel class of c-di-GMP effector proteins in the CRP/FNR superfamily and indicate that XcCLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-GMP concentrations.     

High resolution crystal structure of domain I of the Saccharomyces cerevisiae homing endonuclease PI-SceI

The homing endonuclease PI-SceI from Saccharo myces cerevisiae consists of two domains. The protein splicing domain I catalyzes the excision of the mature endonuclease (intein) from a precursor protein and the religation of the flanking amino acid sequences (exteins) to a functional protein. Furthermore, domain I is involved in binding and recognition of the specific DNA substrate. Domain II of PI-SceI, the endonuclease domain, which is structurally homologous to other homing endonucleases from the LAGLIDADG family, harbors the endonucleolytic center of PI-SceI, which in vivo initiates the homing process by introducing a double-strand cut in the ~35 bp recognition sequence. At 1.35 Å resolution, the crystal structure of PI-SceI domain I provides a detailed view of the part of the protein that is responsible for tight and specific DNA binding. A geometry-based docking of the 75° bent recognition sequence to the full-length protein implies a conformational change or hinge movement of a subdomain of domain I, the tongs part, that is predicted to reach into the major groove near base pairs +16 to +18.     

Structural basis of TRAPPIII‐mediated Rab1 activation

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.