I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548, 14043]
Abstract: Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å co-crystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared to the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein–protein interaction.
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Nov 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Abstract: Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail a3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located > 40 Å apart in the same monomer but adjacent in two neighbouring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in inter-subunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.
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Nov 2020
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B21-High Throughput SAXS
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Abstract: Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV non-structural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.
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Oct 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Abstract: Pectins are a major dietary nutrient source for the human gut microbiota (HGM). The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases (PMEs) essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II-derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric and nuclear magnetic resonance approaches revealed that it is a bi-methylated nona-oligosaccharide GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an alpha/beta (α/β) hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for PMEs, which are predominantly right-handed β-helical proteins. Bioinformatics analyses revealed that the family is dominated by sequences from the prominent genera of the HGM, including Bacteroides and Prevotella. Our results not only highlight the critical role played by this family of enzymes in pectin metabolism but provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.
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Oct 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7641, 9475, 13467]
Abstract: Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, this data provides important new insights to the factors determining stability, substrate recognition and the structural mechanism of hydrolysis in this industrially important group of enzymes.
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Oct 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Abstract: Aminopeptidase N (APN, CD13) is a trans-membrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction, and is involved in the activation of Matrix Metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases such as fibrosis, rheumatoid arthritis, tumor angiogenesis and metastasis as well as viral infections. However, the exact mechanism that leads to APN driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins binding APN induces the transcription of MMPs. As a first step, we sought to identify potential 14‑3‑3 binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3 binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding Fluorescence Polarization (FP) assays and thermodynamic analysis (ITC). In addition, we identified a secondary, non-canonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14‑3‑3σ bound to mono- and bis‑phosphorylated APN derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and opens the field for further investigation of this important signaling pathway.
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Oct 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18666]
Abstract: The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirmed this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. Data are available via ProteomeXchange with identifier PXD021408.
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Sep 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Cecilia
Piergentili
,
Jennifer
Ross
,
Didi
He
,
Kelly J.
Gallagher
,
Will A.
Stanley
,
Laurène
Adam
,
C. Logan
Mackay
,
Arnaud
Basle
,
Kevin J.
Waldron
,
David J.
Clarke
,
Jon
Marles-wright
Diamond Proposal Number(s):
[9487]
Abstract: Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and neighboring secondary metal binding sites. We have previously identified a putative metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.
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Sep 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: Centrioles are key eukaryotic organelles responsible for the formation of cilia and flagella, and for organising the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerisation of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organisation of further organelle components. A dimerisation interaction between SAS-6 N-terminal ‘head’ domains was previously shown to be essential for protein oligomerisation in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low molecular weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerisation. We demonstrate using nuclear magnetic resonance spectroscopy that a ligand from this family binds at the head domain dimerisation site of algae, nematode and human SAS-6 variants, but also that another ligand specifically recognises human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest ligand specificity derives from favourable Van der Waals interactions with a hydrophobic cavity at the dimerisation site.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11265]
Abstract: Ribonucleotide reductase (RNR) is a central enzyme for DNA building block synthesis. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, electron microscopy, x-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral, but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.
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Sep 2020
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