Unusual Conformation of the SxN Motif in the Crystal Structure of Penicillin-Binding Protein A from Mycobacterium tuberculosis

PBPA from Mycobacterium tuberculosis (Mtb) is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in Mtb, but is important for proper cell division in M. smegmatis. We have determined the crystal structure of PBPA at 2.05 Å 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 that characterize 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 disulphide 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 β5 and α11, which restricts the space available in the active site of PBPA, and suggests that conformational changes would be required to accommodate peptide substrate or β-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

The Role of the β5–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 M. tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of the apo enzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-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 second-order acylation rate constants for the antibiotics, imipenem, penicillin G and ceftriaxone. Of these, imipenem, which has demonstrable antitubercular 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 β5-α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that PBPs in apo form can occupy different conformational states

A large displacement of the SXN motif of Cys 115 -modified penicillin-binding protein 5 from Escherichia coli

Penicillin-binding proteins (PBPs), which are the lethal targets of β-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 Å (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

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

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 β2a-β2d 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 β2a-β2d 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 multi-sequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g. by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved

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 β2c−β2d 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 β-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 β-lactam ring during acylation because of the negative charge of its side chain

The RNA chaperon activity of the human La protein (LARP3): DOI: 10.14800/rd.872

Single-stranded RNA molecules fold intensively into secondary and tertiary structures and are often trapped in non-functional configurations. To adapt a functional configuration, structural changes have to be achieved. RNA helicases and RNA chaperones are proteins able to assist those structural rearrangements in an ATP-dependent or ATP-independent manner, respectively. The cancer-associated RNA-binding protein La (LARP3) is an RNA chaperone involved in various aspects of the RNA metabolism. Recently the RNA chaperone domain within the human La protein has been mapped and demonstrated that its activity is required to stimulate cyclin D1-internal ribosome entry site (IRES)-dependent protein synthesis. Furthermore, it has been shown that the La protein can be phosphorylated by serine/threonine kinase AKT in vitro. Taken together, we suggest a model in which the RNA chaperone La stimulates translation of specific target mRNAs by assisting structural changes in their translation start site surrounding RNA region

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

ObjectiveThe 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 methodsWe 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.ResultsWe 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 conclusionSome 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

Structural Effect of the Asp345a Insertion in Penicillin-Binding Protein 2 from Penicillin-Resistant Strains of <i>Neisseria gonorrhoeae</i>

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of <i>Neisseria gonorrhoeae</i> 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 β2c−β2d 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 β-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 β-lactam ring during acylation because of the negative charge of its side chain