Crystal structure of the N‐terminal methyltransferase‐like domain of anamorsin

Anamorsin is a recently identified molecule that inhibits apoptosis during hematopoiesis. It contains an N‐terminal methyltransferase‐like domain and a C‐terminal Fe‐S cluster motif. Not much is known about the function of the protein. To better understand the function of anamorsin, we have solved the crystal structure of the N‐terminal domain at 1.8 Å resolution. Although the overall structure resembles a typical S‐adenosylmethionine (SAM) dependent methyltransferase fold, it lacks one α‐helix and one β‐strand. As a result, the N‐terminal domain as well as the full‐length anamorsin did not show S‐adenosyl‐l ‐methionine (AdoMet) dependent methyltransferase activity. Structural comparisons with known AdoMet dependent methyltransferases reveals subtle differences in the SAM binding pocket that preclude the N‐terminal domain from binding to AdoMet. The N‐terminal methyltransferase‐like domain of anamorsin probably functions as a structural scaffold to inhibit methyl transfers by out‐competing other AdoMet dependant methyltransferases or acts as bait for protein–protein interactions.Proteins 2014; 82:1066–1071. © 2013 Wiley Periodicals, Inc.     

Crystal structure of the PepSY‐containing domain of the YpeB protein involved in germination of bacillus spores

The crystal structure of the C‐terminal domain of the Bacillus megaterium YpeB protein has been solved by X‐ray crystallography to 1.80‐Å resolution. The full‐length protein is essential in stabilising the SleB cortex lytic enzyme in Bacillus spores, and may have a role in regulating SleB activity during spore germination. The YpeB‐C crystal structure comprises three tandemly repeated PepSY domains, which are aligned to form an extended laterally compressed molecule. A predominantly positively charged region located in the second PepSY domain may provide a site for protein interactions that are important in stabilising SleB and YpeB within the spore. Proteins 2015; 83:1914–1921. © 2015 Wiley Periodicals, Inc.     

Crystal structure of the N‐terminal domain of EccA1 ATPase from the ESX‐1 secretion system of Mycobacterium tuberculosis

EccA₁ is an important component of the type VII secretion system (T7SS) that is responsible for transport of virulence factors in pathogenic mycobacteria. EccA₁ has an N‐terminal domain of unknown function and a C‐terminal AAA+ (ATPases associated with various cellular activities) domain. Here we report the crystal structure of the N‐terminal domain of EccA₁ from Mycobacterium tuberculosis, which shows an arrangement of six tetratricopeptide repeats that may mediate interactions of EccA₁ with secreted substrates. Furthermore, the size and shape of the N‐terminal domain suggest its orientation in the context of a hexamer model of full‐length EccA₁. Proteins 2014; 82:159–163. © 2013 Wiley Periodicals, Inc.     

Crystal structure of human Ankyrin G death domain

Ankyrins (Ank) are a ubiquitously expressed family of multifunctional membrane adapter proteins. Ankyrin G (AnkG) is critical for assembling and maintenance of the axon initial segment. Here we present the 2.1 Å crystal structure of human AnkG death domain (hAnkG‐DD). The core death domain is composed of six α‐helices and three 3₁₀‐helices. It forms a hydrophobic pocket on the surface of the molecule. The C‐terminal tail of the hAnkG‐DD curves back to have the aromatic ring of a phenylalanine residue, Phe100 insert into this pocket, which anchors the flexible tail onto the core domain. Related DDs were selected for structure comparison. The major variations are at the C‐terminal region, including the α6 and the long C‐terminal extension. The results of size exclusion chromatography and analytical ultracentrifugation suggest that hAnkG‐DD exists as monomer in solution. Our work should help for the future investigation of the structure–function of AnkG. Proteins 2014; 82:3476–3482. © 2014 Wiley Periodicals, Inc.     

Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β‐barrel core domain resembling type VI secretion system proteins and a two‐helix pair

The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5‐Å resolution. The YwpF structure consists of two regions: an N‐terminal core β‐barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C‐terminal two‐helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. Proteins 2015; 83:781–788. © 2015 Wiley Periodicals, Inc.     

Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase‐1

The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N‐terminal pyrin domain (PYD) and a C‐terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase‐1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose‐binding protein. The structure reveals the six‐helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase‐1 CARD model suggests potential mechanisms for their association through electrostatic attraction. Proteins 2013; 81:1266–1270. © 2013 Wiley Periodicals, Inc.     

Crystal Structure Confirmation of JHP933 as a Nucleotidyltransferase Superfamily Protein from Helicobacter pylori Strain J99

Helicobacter pylori is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of H. pylori strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αβαβαβα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.     

The macro domain as fusion tag for carrier‐driven crystallization

Obtaining well‐ordered crystals remains a significant challenge in protein X‐ray crystallography. Carrier‐driven crystallization can facilitate crystal formation and structure solution of difficult target proteins. We obtained crystals of the small and highly flexible SPX domain from the yeast vacuolar transporter chaperone 4 (Vtc4) when fused to a C‐terminal, non‐cleavable macro tag derived from human histone macroH2A1.1. Initial crystals diffracted to 3.3 Å resolution. Reductive protein methylation of the fusion protein yielded a new crystal form diffracting to 2.1 Å. The structures were solved by molecular replacement, using isolated macro domain structures as search models. Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier‐driven crystallization.     

Substrate‐mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N‐acetylneuraminic acid synthase

N‐Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesized in prokaryotes via a condensation reaction between phosphoenolpyruvate and N‐acetylmannosamine. This reaction is catalyzed by a domain swapped, homodimeric enzyme, N‐acetylneuraminic acid synthase (NANAS). NANAS comprises two distinct domains; an N‐terminal catalytic (β/α)₈ barrel linked to a C‐terminal antifreeze protein‐like (AFPL) domain. We have investigated the role of the AFPL domain by characterizing a truncated variant of NmeNANAS, which was discovered to be soluble yet inactive. Analytical ultracentrifugation and analytical size exclusion were used to probe the quaternary state of the NmeNANAS truncation, and revealed that loss of the AFPL domain destabilizes the dimeric form of the enzyme. The results from this study thereby demonstrate that the AFPL domain plays a critical role for both the catalytic function and quaternary structure stability of NANAS. Small angle X‐ray scattering, molecular dynamics simulations, and amino acid substitutions expose a complex hydrogen‐bonding relay, which links the roles of the catalytic and AFPL domains across subunit boundaries. Proteins 2014; 82:2054–2066. © 2014 Wiley Periodicals, Inc.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

Sufu (Suppressor of Fused), a two‐domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu‐like proteins have previously been identified based on sequence similarity to the N‐terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu‐like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu‐like protein. The structure revealed a striking similarity to the N‐terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ～15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu‐like proteins that are present in ～200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.     

The crystal structure of Clostridium perfringens SleM,a muramidase involved in cortical hydrolysis during spore germination

Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the C. perfringens S40 SleM protein at 1.8 Å. SleM comprises an N‐terminal catalytic domain that adopts an irregular α/β‐barrel fold that is common to GH25 family lysozymes, plus a C‐terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Proteins 2016; 84:1681–1689. © 2016 Wiley Periodicals, Inc.     

Crystal structure of the N domain of Lon protease from Mycobacterium avium complex

Lon protease is evolutionarily conserved in prokaryotes and eukaryotic organelles. The primary function of Lon is to selectively degrade abnormal and certain regulatory proteins to maintain the homeostasis in vivo. Lon mainly consists of three functional domains and the N‐terminal domain is required for the substrate selection and recognition. However, the precise contribution of the N‐terminal domain remains elusive. Here, we determined the crystal structure of the N‐terminal 192‐residue construct of Lon protease from Mycobacterium avium complex at 2.4 å resolution,and measured NMR‐relaxation parameters of backbones. This structure consists of two subdomains, the β‐strand rich N‐terminal subdomain and the five‐helix bundle of C‐terminal subdomain, connected by a flexible linker,and is similar to the overall structure of the N domain of Escherichia coli Lon even though their sequence identity is only 26%. The obtained NMR‐relaxation parameters reveal two stabilized loops involved in the structural packing of the compact N domain and a turn structure formation. The performed homology comparison suggests that structural and sequence variations in the N domain may be closely related to the substrate selectivity of Lon variants. Our results provide the structure and dynamics characterization of a new Lon N domain, and will help to define the precise contribution of the Lon N‐terminal domain to the substrate recognition.     

Crystal structure of the protein At3g01520, a eukaryotic universal stress protein‐like protein from arabidopsis thaliana in complex with AMP

Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain‐containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N‐terminal portion of a multi‐domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP‐like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single‐wavelength anomalous dispersion method and refined to an R factor of 21.8% (R_free = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann‐like α/β overall fold. The bound AMP and conservation of residues in the ATP‐binding loop suggest that the protein At3g01520 also belongs to the ATP‐binding USP subfamily members. Proteins 2015; 83:1368–1373. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Apo raver1 structure reveals distinct RRM domain orientations

Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and α‐actinin. The amino‐terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1–3 domains in complex with Vt at 2.75 Å resolution. Here, we report crystal structure of the unbound raver1 RRM1–3 domains at 2 Å resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30°. Superposition with other RNA‐bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three‐tandem RRM domain structure.     

Crystal structure of BinB: A receptor binding component of the binary toxin from Lysinibacillus sphaericus

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N‐ and C‐termini. The globular N‐terminal domain has a β‐trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor‐binding. The BinB β‐rich C‐terminal domain shares similar three‐dimensional folding with aerolysin type β‐pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin‐like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation. Proteins 2014; 82:2703–2712. © 2014 Wiley Periodicals, Inc.