Mapping of UK Biobank clinical codes: Challenges and possible solutions.

OBJECTIVE: The UK Biobank provides a rich collection of longitudinal clinical data coming from different healthcare providers and sources in England, Wales, and Scotland. Although extremely valuable and available to a wide research community, the heterogeneous dataset contains inconsistent medical terminology that is either aligned to several ontologies within the same category or unprocessed. To make these data useful to a research community, data cleaning, curation, and standardization are needed. Significant efforts to perform data reformatting, mapping to any selected ontologies (such as SNOMED-CT) and harmonization are required from any data user to integrate UK Biobank hospital inpatient and self-reported data, data from various registers with primary care (GP) data. The integrated clinical data would provide a more comprehensive picture of one's medical history. MATERIALS AND METHODS: We evaluated several approaches to map GP clinical Read codes to International Classification of Diseases (ICD) and Systematized Nomenclature of Medicine Clinical Terms (SNOMED CT) terminologies. The results were compared, mapping inconsistencies were flagged, a quality category was assigned to each mapping to evaluate overall mapping quality. RESULTS: We propose a curation and data integration pipeline for harmonizing diagnosis. We also report challenges identified in mapping Read codes from UK Biobank GP tables to ICD and SNOMED CT. DISCUSSION AND CONCLUSION: Some of the challenges-the lack of precise one-to-one mapping between ontologies or the need for additional ontology to fully map terms-are general reflecting trade-offs to be made at different steps. Other challenges are due to automatic mapping and can be overcome by leveraging existing mappings, supplemented with automated and manual curation.

Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics.

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the ß3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the ß3-ß4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells.

The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.

Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness.

Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upward of 60 amino acid changes relative to wild-type PBP2, or by nonmosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates that harbored a mosaic penA allele with an A501P mutation were identified. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon 501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, and A501S) that increased resistance and preserved essential transpeptidase function were isolated. To understand their structural implications, these mutations were introduced into the nonmosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was determined and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation.

The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.

Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the ß2c-ß2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the ß-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the ß-lactam ring during acylation because of the negative charge of its side chain.

High-throughput screening for novel inhibitors of Neisseria gonorrhoeae penicillin-binding protein 2.

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of ß-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for ß-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.

A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the ß2a-ß2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the ß2a-ß2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by ß-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

The role of the ß5-α11 loop in the active-site dynamics of acylated penicillin-binding protein A from Mycobacterium tuberculosis.

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a ß-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the ß5-α11 loop near the active site, but these differ for each ß-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the ß5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.

Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis.

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 A resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characteristic of penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the "x" of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between beta 5 and alpha 11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or beta-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.

Crystal structure of CD59: implications for molecular recognition of the complement proteins C8 and C9 in the membrane-attack complex.

Human CD59 is a small membrane-bound glycoprotein that functions as an inhibitor of the membrane-attack complex (MAC) of the complement system by binding the complement proteins C8 and C9. The crystal structure of a soluble construct of CD59 has been determined to 2.1 A resolution. When compared with previous models of CD59 determined using NMR, some interesting differences are noted, including the position of helix alpha1, which contributes to the binding surface for C8 and C9. Interestingly, the crystal structure superimposes more closely with an updated NMR model of CD59 that was produced using Monte Carlo minimization, including helix alpha1. Mapping of mutations associated with enhanced or lowered inhibitory function of CD59 show the binding region to be located in a crevice between alpha1 and a three-stranded beta-sheet, as has been identified previously. Residues in the core of this region are well ordered in the electron density, in part owing to a network of stabilizing covalent and noncovalent interactions, and manifest an interesting 'striped' distribution of hydrophobic and basic residues. Docking of the same peptide that was modeled previously into the NMR structure shows that Arg55, which has been postulated to exist in 'open' and 'closed' positions, is intermediate in position between these two and is well placed to contact the peptide. Further clues regarding how CD59 interacts with small peptides arise from the crystal packing of this structure, which shows that a symmetry-related loop comprising residues 20-24 occupies a spatially similar position to the modeled peptide. This higher resolution structure of CD59 will facilitate a more precise dissection of its interactions with C8 and C9 and thus increase the likelihood of designing enhanced CD59-based therapeutics.

Structure of the N-terminal domain of human CEACAM1: binding target of the opacity proteins during invasion of Neisseria meningitidis and N. gonorrhoeae.

CEACAM1 is a cellular adhesion molecule whose protein expression is down-regulated in several carcinomas and which also contributes to the pathogenicity of Neisseria by acting as a receptor for Opa proteins. The crystal structure of the N-terminal (D1) domain of human CEACAM1 has been determined at 2.2 Angstrom resolution. The structure shows several differences compared with a lower resolution model of the same domain from mouse solved previously, especially in the functional regions. Mapping of the sites of mutations that lower or abolish the binding of CEACAM1 to Opa proteins shows a distinct clustering of residues on the GFCC'C'' face of the molecule. Prominent amongst these are residues in the C, C' and F strands and the CC' loop. A similar analysis shows that the region responsible for homophilic or heterophilic interactions of CEACAM1 is also on the GFCC'C'' face and overlaps partially with the Opa-binding region. This higher resolution structure of CEACAM1 will facilitate a more precise dissection of its functional regions in the context of neisserial pathogenesis, cellular adhesion and immune evasion.

A large displacement of the SXN motif of Cys115-modified penicillin-binding protein 5 from Escherichia coli.

Penicillin-binding proteins (PBPs), which are the lethal targets of beta-lactam antibiotics, catalyse the final stages of peptidoglycan biosynthesis of the bacterial cell wall. PBP 5 of Escherichia coli is a D-alanine CPase (carboxypeptidase) that has served as a useful model to elucidate the catalytic mechanism of low-molecular-mass PBPs. Previous studies have shown that modification of Cys115 with a variety of reagents results in a loss of CPase activity and a large decrease in the rate of deacylation of the penicilloyl-PBP 5 complex [Tamura, Imae and Strominger (1976) J. Biol. Chem. 251, 414-423; Curtis and Strominger (1978) J. Biol. Chem. 253, 2584-2588]. The crystal structure of wild-type PBP 5 in which Cys115 fortuitously had formed a covalent adduct with 2-mercaptoethanol was solved at 2.0 A (0.2 nm) resolution, and these results provide a structural rationale for how thiol-directed reagents lower the rate of deacylation. When compared with the structure of the unmodified wild-type enzyme, a major change in the architecture of the active site is observed. The two largest differences are the disordering of a loop comprising residues 74-90 and a shift in residues 106-111, which results in the displacement of Ser110 of the SXN active-site motif. These results support the developing hypothesis that the SXN motif of PBP 5, and especially Ser110, is intimately involved in the catalytic mechanism of deacylation.