The crystal structure analysis of group B Streptococcus sortase C1: a model for the "lid" movement upon substrate binding

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible “lid” anchored in the active site. However, the mechanistic details of the “lid” displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its “lid” mutant and proposed a role of the “lid” as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.     

Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond

A recombinant Fv construct of the B1 monoclonal antibody that recognizes the Lewis^Y-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the V_H and V_L domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues V_L100 and V_H44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023–1029, 1995). The B1 disulfide-stabilized Fv (B1dsFv) crystallizes in space group P6₁22 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1-Å resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10-Å wide and 17-Å long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the Lewis^Y tetrasaccharide, Fuc–Gal–Nag–Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781–3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab. Proteins 31:128–138, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.     

MQAPsingle: A quasi single‐model approach for estimation of the quality of individual protein structure models

We present a Model Quality Assessment Program (MQAP), called MQAPsingle, for ranking and assessing the absolute global quality of single protein models. MQAPsingle is quasi single‐model MQAP, a method that combines advantages of both “pure” single‐model MQAPs and clustering MQAPs. This approach results in higher accuracy compared to the state‐of‐the‐art single‐model MQAPs. Notably, the prediction for a given model is the same regardless if this model is submitted to our server alone or together with other models. Proteins 2016; 84:1021–1028. © 2015 Wiley Periodicals, Inc.     

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.     

The structure of the PP2A regulatory subunit B56 gamma: the remaining piece of the PP2A jigsaw puzzle

The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56 gamma. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56 gamma and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56 gamma first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly.     

The crystal structure of a binary complex of two pseudopilins: EpsI and EpsJ from the type 2 secretion system of Vibrio vulnificus

Type II secretion systems (T2SS) translocate virulence factors from the periplasmic space of many pathogenic bacteria into the extracellular environment. The T2SS of Vibrio cholerae and related species is called the extracellular protein secretion (Eps) system that consists of a core of multiple copies of 11 different proteins. The pseudopilins, EpsG, EpsH, EpsI, EpsJ and EpsK, are five T2SS proteins that are thought to assemble into a pseudopilus, which is assumed to interact with the outer membrane pore, and may actively participate in the export of proteins. We report here biochemical evidence that the minor pseudopilins EpsI and EpsJ from Vibrio species interact directly with one another. Moreover, the 2.3 Å resolution crystal structure of a complex of EspI and EpsJ from Vibrio vulnificus represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS. Both EpsI and EpsJ appear to be structural extremes within the family of type 4a pilin structures solved to date, with EpsI having the smallest, and EpsJ the largest, “variable pilin segment” seen thus far. A high degree of sequence conservation in the EpsI:EpsJ interface indicates that this heterodimer occurs in the T2SS of a large number of bacteria. The arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helical character of proteins assembled into a pseudopilus.     

X-ray structure of a two-domain type laccase: A missing link in the evolution of multi-copper proteins

A multi-copper protein with two cupredoxin-like domains was identified from our in-house metagenomic database. The recombinant protein, mgLAC, contained four copper ions/subunits, oxidized various phenolic and non-phenolic substrates, and had spectroscopic properties similar to common laccases. X-ray structure analysis revealed a homotrimeric architecture for this enzyme, which resembles nitrite reductase (NIR). However, a difference in copper coordination was found at the domain interface. mgLAC contains a T2/T3 tri-nuclear copper cluster at this site, whereas a mononuclear T2 copper occupies this position in NIR. The trimer is thus an essential part of the architecture of two-domain multi-copper proteins, and mgLAC may be an evolutionary precursor of NIR.     

A structure prediction for the ligand-binding region of the integrin β subunit: evidence for the presence of a von Willebrand factor A domain

The integrins are a family of cell surface receptors that mediate biologically important adhesive interactions. Integrin-ligand binding has been extensively studied because of the potential for the development of anti-adhesive therapies, but the molecular basis of this interaction is still poorly understood. A conserved region near the N-terminus of the β subunit appears to be of particular importance in ligand binding, but to date this domain has not been expressed in isolation. As a prelude to expression and potential structure determination, we have performed a detailed structure prediction for this region. Primary, secondary and tertiary structure analyses indicate that the region folds into a von Willebrand factor A-domain, thereby potentially placing a previously characterised module at the centre of a key functional region.     

A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure

An updated topological model is constructed for the catalytic nucleotide-binding site of the F₁-ATPase. The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and theras p21 protein. Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the subunit of F₁. Several potentially important residues on are noted that have not yet been studied by mutagenesis or affinity labeling.     

A model for the structure of a homodimeric prohormone: The precursor to the locust neuropeptide AKH I

We have determined the structure in solution of a homodimeric protein that is a precursor to the locust neuropeptide adipokinetic hormone I using nuclear magnetic resonance spectroscopy. This precursor, called P1, is comprised of two 41 residue strands joined by a single inter-chain disulphide at Cys39. We have also determined the structure of an end product of P1 processing, called APRP1; this is a homodimer comprised of residues 14–41 of PI. Nuclear Overhauser Effect (nOe) data indicate that in both P1 and APRP1, residues 22–37 (numbered with respect to P1) form pairs of α-helices, with no evidence for any other secondary structure. © 1994 Wiley-Liss, Inc.     

B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes

The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology.     

High-resolution structure of the stable plasminogen activator inhibitor type-1 variant 14-1B in its proteinase-cleaved form: a new tool for detailed interaction studies and modeling

Wild-type plasminogen activator inhibitor type-1 (PAI-1) rapidly converts to the inactive latent state under conditions of physiological pH and temperature. For in vivo studies of active PAI-1 in cell culture and in vivo model systems, the 14-1B PAI-1 mutant (N150H-K154T-Q319L-M354I), with its stabilized active conformation, has thus become the PAI-1 of choice. As a consequence of the increased stability, the only two forms likely to be encountered are the active or the cleaved form, the latter either free or complexed with target proteinase. We hereby report the first structure of the stable 14-1B PAI-1 variant in its reactive center cleaved form, to a resolution of 2.0 A. The >99% complete structure represents the highest resolved structure of free cleaved PAI-1. This high-resolution structure should be of great use for drug target development and for modeling protein-protein interactions such as those of PAI-1 with vitronectin.     

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target

The binding process of A9 peptide toward HER2‐DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2‐DIVMP, multiple ligand‐based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.     

The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function

Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the β-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 °C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the β-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.     

Lock and chop: A novel method for the generation of a PICK1 PDZ domain and piperidine‐based inhibitor co‐crystal structure

The membrane protein interacting with kinase C1 (PICK1) plays a trafficking role in the internalization of neuron receptors such as the amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor. Reduction of surface AMPA type receptors on neurons reduces synaptic communication leading to cognitive impairment in progressive neurodegenerative diseases such as Alzheimer disease. The internalization of AMPA receptors is mediated by the PDZ domain of PICK1 which binds to the GluA2 subunit of AMPA receptors and targets the receptor for internalization through endocytosis, reducing synaptic communication. We planned to block the PICK1‐GluA2 protein–protein interaction with a small molecule inhibitor to stabilize surface AMPA receptors as a therapeutic possibility for neurodegenerative diseases. Using a fluorescence polarization assay, we identified compound BIO124 as a modest inhibitor of the PICK1‐GluA2 interaction. We further tried to improve the binding affinity of BIO124 using structure‐aided drug design but were unsuccessful in producing a co‐crystal structure using previously reported crystallography methods for PICK1. Here, we present a novel method through which we generated a co‐crystal structure of the PDZ domain of PICK1 bound to BIO124.