Crystal structure of native Anopheles gambiae Serpin-2, a negative regulator of melanization in mosquitoes

Serpins are the dominant group of protease inhibitors in metazoans that control a wide variety of biological processes including major innate immune reactions. One of these inhibitors, SRPN2, controls melanization in mosquitoes – a powerful, arthropod-specific innate immune response. SRPN2 depletion from the hemolymph of adult female mosquitoes significantly reduces longevity and therefore this serpin is a potential target for novel insecticides. We report here the crystal structure of SRPN2 in its native conformation from the African malaria mosquito, Anopheles gambiae to 1.75 Å resolution. SRPN2 adopts a similar fold as observed for other serpins with a core of three β-sheets surrounded by nine α-helices with an exposed reactive center loop (RCL) that extends from the protein body. Similar to other native serpin structures, several residues within the reactive center loop were disordered and could not be modeled. Intriguingly, the N-terminal hinge of the RCL in SRPN2 was found to be inserted into β-sheet A, suggesting a potential activation mechanism analogous to heparin-mediated activation of Antithrombin III

Chlamydia trachomatis CT771 (nudH) is an asymmetric Ap4A hydrolase

Asymmetric diadenosine 5′,5′″-P1,P4-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologs to CT771 within environmental Chlamydiales that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 Å and 1.9 Å resolution, respectively. The structure of CT771 shows a αβα-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap4A hydrolase from Caenorhabditis elegans. These structures represent only the second Ap4A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap4A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap4A hydrolase (nudH)

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site

Copyright © 2018 American Chemical Society. Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role

Structure of the Yersinia pestis tip protein LcrV refined to 1.65 A resolution

This is the publisher's version, also available electronically from http://scripts.iucr.org/cgi-bin/paper?S1744309113008579.The human pathogen Yersinia pestis requires the assembly of the type III secretion system (T3SS) for virulence. The structural component of the T3SS contains an external needle and a tip complex, which is formed by LcrV in Y. pestis. The structure of an LcrV triple mutant (K40A/D41A/K42A) in a C273S background has previously been reported to 2.2 Å resolution. Here, the crystal structure of LcrV without the triple mutation in a C273S background is reported at a higher resolution of 1.65 Å. Overall the two structures are similar, but there are also notable differences, particularly near the site of the triple mutation. The refined structure revealed a slight shift in the backbone positions of residues Gly28-Asn43 and displayed electron density in the loop region consisting of residues Ile46-Val63, which was disordered in the original structure. In addition, the helical turn region spanning residues Tyr77-Gln95 adopts a different orientation

MinD-dependent conformational changes in MinE required for the Min oscillator to spatially regulate cytokinesis

MinD recruits MinE to the membrane leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring in E. coli. How these proteins interact, however, is not clear since the MinD binding regions of MinE are sequestered within a 6-stranded β-sheet and masked by N-terminal helices. Here, minE mutations are isolated that restore interaction to some MinD and MinE mutants. These mutations alter the MinE structure releasing the MinD binding regions and N-terminal helices that bind MinD and the membrane, respectively. Crystallization of MinD-MinE complexes reveals a 4-stranded β-sheet MinE dimer with the released β strands (MinD binding regions) converted to α-helices bound to MinD dimers. These results suggest a 6 stranded, β-sheet dimer of MinE ‘senses’ MinD and switches to a 4-stranded β-sheet dimer that binds MinD and contributes to membrane binding. Also, the results indicate how MinE persists at the MinD-membrane surface

MinD-dependent conformational changes in MinE required for the Min oscillator to spatially regulate cytokinesis

MinD recruits MinE to the membrane leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring in E. coli. How these proteins interact, however, is not clear since the MinD binding regions of MinE are sequestered within a 6-stranded β-sheet and masked by N-terminal helices. Here, minE mutations are isolated that restore interaction to some MinD and MinE mutants. These mutations alter the MinE structure releasing the MinD binding regions and N-terminal helices that bind MinD and the membrane, respectively. Crystallization of MinD-MinE complexes reveals a 4-stranded β-sheet MinE dimer with the released β strands (MinD binding regions) converted to α-helices bound to MinD dimers. These results suggest a 6 stranded, β-sheet dimer of MinE ‘senses’ MinD and switches to a 4-stranded β-sheet dimer that binds MinD and contributes to membrane binding. Also, the results indicate how MinE persists at the MinD-membrane surface

The Hemophore HasA from Yersinia pestis (HasAyp) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change

Hemophores from Serratia marcescens (HasAsm) and Pseudomonas aeruginosa (HasAp) bind hemin between two loops, which harbor the axial ligands H32 and Y75. Hemin binding to the Y75 loop triggers closing of the H32 loop and enables binding of H32. Because Yersinia pestis HasA (HasAyp) presents a Gln at position 32, we determined the structures of apo-and holo-HasAyp. Surprisingly, the Q32 loop in apo-HasAyp is already in the closed conformation but no residue from the Q32 loop binds hemin in holo-HasAyp. In agreement with the minimal reorganization between the apo-and holo-structures, the hemin on-rate is too fast to detect by conventional stopped-flow measurements

Concerted Motions Networking Pores and Distant Ferroxidase Centers Enable Bacterioferritin Function and Iron Traffic

X-ray crystallography, molecular dynamics (MD) simulations and biochemistry were utilized to investigate the effect of introducing hydrophobic interactions in the 4-fold (N148L and Q151L) and B-pores (D34F) of Pseudomonas aeruginosa bacterioferritin B (BfrB) on BfrB function. The structures show only local structural perturbations and confirm the anticipated hydrophobic interactions. Surprisingly, structures obtained after soaking crystals in Fe2+-containing crystallization solution revealed that although iron loads into the ferroxidase centers of the mutants, the side chains of ferroxidase ligands E51 and H130 do not reorganize to bind the iron ions, as is seen in the wt BfrB structures. Similar experiments with a double mutant (C89S/K96C) prepared to introduce changes outside the pores show competent ferroxidase centers that function akin to those in wt BfrB. MD simulations comparing wt BfrB with the D34F and N148L mutants show that the mutants exhibit significantly reduced flexibility, and reveal a network of concerted motions linking ferroxidase centers and 4-fold and B-pores, which are important for imparting ferroxidase centers in BfrB with the required flexibility to function efficiently. In agreement, the efficiency of Fe2+ oxidation and uptake of the 4-fold and B-pore mutants in solution is significantly compromised relative to wt or C89S/K96C BfrB. Finally, our structures show a large number of previously unknown iron binding sites in the interior cavity and B-pores of BfrB, which reveal in unprecedented detail conduits followed by iron and phosphate ions across the BfrB shell, as well as paths in the interior cavity that may facilitate nucleation of the iron phosphate mineral

Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway

BACKGROUND: Specific pathways and components for respiration in Chlamydia are poorly understood. RESULTS: The C. trachomatis hypothetical protein CT263 crystal structure displays strong structural similarity with 5′-methylthioadenosine nucleosidase enzymes. CONCLUSION: Bioinformatic analyses and enzymatic characterization of CT263 suggest menaquinone biosynthesis proceeds through the futalosine pathway in Chlamydiaceae. SIGNIFICANCE: Unique structural aspects of the CT263 active site can be leveraged to modify existing transition state inhibitors

Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.Diphtheria toxin, an exotoxin secreted by Corynebacterium that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5–6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2–3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry.National Institute of General Medical Sciences (P30 GM110761)Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH1135