I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.
|
Mar 2021
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[23269]
Abstract: The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4′-phosphopantetheine, 4′-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker–ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein–protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.
|
Jan 2021
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[25819]
Abstract: Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and non‐reducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP2 and ACP1‐ACP2)–thioesterase (TE) domains from NR‐PKS involved in menisporopsin A biosynthesis. Small angle X‐ray scattering (SAXS) studies in combination with homology modelling reveal that these polypeptides adopt a distinctive Beads‐on‐a‐String configuration, characterized by the presence of highly flexible interdomain linkers. These models provide a platform for studying domain organization and interdomain interactions in fungal NR‐PKSs, which may be of value in directing the design of functionally optimised polyketide scaffolds.
|
Oct 2020
|
|
B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Alice J.
Bochel
,
Christopher
Williams
,
Airlie J.
Mccoy
,
Hans-Jürgen
Hoppe
,
Ashley J.
Winter
,
Ryan D.
Nicholls
,
Karl
Harlos
,
E. Yvonne
Jones
,
Imre
Berger
,
A. Bassim
Hassan
,
Matthew P.
Crump
Diamond Proposal Number(s):
[8423]
Abstract: The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7–10 within two multi-domain constructs, D9–10 and D7–11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7–11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7–11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.
|
Sep 2020
|
|
B21-High Throughput SAXS
|
Catherine R.
Back
,
Victoria A.
Higman
,
Kristian
Levay
,
Viren V.
Patel
,
Alice E.
Parnell
,
Daniel
Frankel
,
Howard F.
Jenkinson
,
Steven G.
Burston
,
Matthew P.
Crump
,
Angela H.
Nobbs
,
Paul R.
Race
Diamond Proposal Number(s):
[13400, 12342]
Open Access
Abstract: The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to seventeen ~100-amino-acid-long repeat domains (RDs). Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1–17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all β-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.
|
Mar 2020
|
|
I04-Macromolecular Crystallography
|
Christopher D.
Fage
,
Thomas
Lathouwers
,
Michiel
Vanmeert
,
Ling-Jie
Gao
,
Kristof
Vrancken
,
Eveline-Marie
Lammens
,
Angus
Weir
,
Ruben
Degroote
,
Harry
Cuppens
,
Simone
Kosol
,
Thomas J.
Simpson
,
Matthew P.
Crump
,
Christine L.
Willis
,
Piet
Herdewijn
,
Eveline
Lescrinier
,
Rob
Lavigne
,
Jozef
Anné
,
Joleen
Masschelein
Diamond Proposal Number(s):
[14692]
Abstract: The enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus . By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development.
|
Mar 2020
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[12342]
Abstract: Oxygen heterocycles—in particular, tetrahydropyrans (THPs) and tetrahydrofurans—are common structural features of many biologically active polyketide natural products. Mupirocin is a clinically important antibiotic isolated from Pseudomonas fluorescens and is assembled on a THP ring, which is essential for bioactivity. However, the biosynthesis of this moiety has remained elusive. Here, we show an oxidative enzyme-catalysed cascade that generates the THP ring of mupirocin. Rieske non-haem oxygenase (MupW)-catalysed selective oxidation of the C8–C16 single bond in a complex acyclic precursor is combined with an epoxide hydrolase (MupZ) to catalyse the subsequent regioselective ring formation to give the hydroxylated THP. In the absence of MupZ, a five-membered tetrahydrofuran ring is isolated, and model studies are consistent with cyclization occurring via an epoxide intermediate. High-resolution X-ray crystallographic studies, molecular modelling and mutagenesis experiments of MupZ provide insights into THP ring formation proceeding via an anti-Baldwin 6-endo-tet cyclization.
|
Dec 2018
|
|
B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Ashley J.
Winter
,
Christopher
Williams
,
Michail N.
Isupov
,
Hannah
Crocker
,
Mariya
Gromova
,
Philip
Marsh
,
Oliver J.
Wilkinson
,
Mark S.
Dillingham
,
Nicholas J.
Harmer
,
Richard W.
Titball
,
Matthew P.
Crump
Diamond Proposal Number(s):
[12342]
Open Access
Abstract: Toxin–antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB–DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution respectively and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a hetero-octameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of hicAB in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex.These results reveal the structural mechanisms by which HicA de-represses the HicB–DNA complex.
|
Oct 2018
|
|
I04-Macromolecular Crystallography
|
Susana
Frago
,
Ryan D.
Nicholls
,
Madeleine
Strickland
,
Jennifer
Hughes
,
Christopher
Williams
,
Lee
Garner
,
Mirvat
Surakhy
,
Rory
Maclean
,
Dellel
Rezgui
,
Stuart N.
Prince
,
Oliver J.
Zaccheo
,
Daniel
Ebner
,
Sabina
Sanegre
,
Sheng
Yu
,
Francesca M.
Buffa
,
Matthew P.
Crump
,
Andrew Bassim
Hassan
Diamond Proposal Number(s):
[8922]
Abstract: Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.
|
May 2016
|
|
I02-Macromolecular Crystallography
|
Abstract: The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.
|
May 2011
|
|
