Biology subjects

Meek, R. W.

Publications and source records attributed to Meek, R. W..

5 recordsLinked to original sources

bioRxiv2023

Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is therefore regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of [~]108 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a non-competitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest an unprecedented mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity. SIGNIFICANCE STATEMENTThousands of proteins within humans are modified by the monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAc regulates cellular physiology and is being pursued to create therapeutics. Remarkably, only one enzyme, O-GlcNAc transferase (OGT), installs O-GlcNAc and its regulation is poorly understood. By affinity selection using a vast peptide library, we uncover an amino acid sequence motif that drives binding of polypeptides to OGT. An OGT-peptide complex shows how this motif binds to an allosteric site proximal to the active site and inhibits OGT in an unprecedented manner. Given the distribution of this sequence motif within the human proteome proteins containing this motif likely regulate the activity of OGT, outlining a new mode by which OGT is controlled and opening new avenues for research.

biochemistry↗

bioRxiv2023

Sulfolactaldehyde dehydrogenase of Rhizobium leguminosarum: Kinetic and structural analysis

Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle. Graphical abstractSulfolactate is an important species in the biogeochemical sulfur cycle. Herein we report the 3D cryo-EM structure and kinetics of its biosynthetic enzyme, sulfolactaldehyde dehydrogenase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/532361v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f3d1d3org.highwire.dtl.DTLVardef@548615org.highwire.dtl.DTLVardef@12efc0eorg.highwire.dtl.DTLVardef@1a6145c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Structures of Bdellovibrio bacteriovorus phosphoglucose isomerase reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding.

Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI) which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate (F6P). The predatory bacterium Bdellovibrio bacteriovorus leads a complex lifecycle, switching between intraperiplasmic replicative and extracellular "hunter" attack-phase stages. Passage through this complex lifecycle involves different metabolic states. Here we present the unliganded and substrate bound structures of the Bdellovibrio bacteriovorus PGI, solved to 1.74[A] and 1.67 [A], respectively. These structures reveal that an induced-fit conformational change within the active site is not a pre-requisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for a glycine in Bdellovibrio and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterised to date and likely represents the minimal requirements for a functional PGI.

biochemistry↗

bioRxiv2020

Structure-function characterization of the conserved regulatory mechanism of the Escherichia coli M48 metalloprotease BepA

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 [A], in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. ImportanceM48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the LPS biogenesis machinery. Here we present the structure of BepA and the results of a structure guided mutagenesis strategy, which reveal the key residues required for activity.

microbiology↗

bioRxiv2019

Structure-function analysis of the Escherichia coli β-barrel assembly enhancing protease BepA suggests a role for a self-inhibitory state

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for the bacteria against environmental insults and attack by antimicrobials. The key component of the OM barrier is the surface exposed lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system OM component, LptD, is essential and is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA, solved to a resolution of 1.9 [A]. Our structure comprises the zinc-bound m48 protease domain and a tetratricopeptide repeat (TPR) domain, consisting of four 2-helix TPR motifs and four non-TPR helices, leading to a nautilus-like shape in which the TPR repeats cup the protease domain. Using targeted mutagenesis approaches, we demonstrate that the protein is auto-regulated by the active-site plug. Further to this we reveal that mutation of a negative pocket, formed at the interface between the m48 and TPR domains, impairs BepA activity suggesting the pocket as a possible substrate binding site. We also identify a potential protein interaction site within the TPR cavity as being important for BepA function. Lastly, we provide evidence to show that increased antibiotic susceptibility in the absence of correctly functioning BepA occurs through disruption of OM lipid asymmetry, leading to reduced barrier function and increased cell permeability.

microbiology↗