I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[15916, 21426]
Open Access
Abstract: The type IIB receptor protein tyrosine phosphatases (R2B RPTPs) are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of R2B RPTPs form stable, homophilic, trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms head-to-tail homodimers. However, as the interface was composed of residues conserved across the family, the determinants of homophilic specificity remain unknown. Here, we have solved the X-ray crystal structure of the membrane-distal, N-terminal domains of PTPRK that form a head-to-tail dimer consistent with intermembrane adhesion. Comparison with the PTPRM structure demonstrates inter-domain conformational differences that may define homophilic specificity. Using small-angle X-ray scattering, we determined the solution structures of the full-length ECDs of PTPRM and PTPRK, identifying that both are rigid, extended molecules that differ in their overall long-range conformation. Furthermore, we identify one residue, W351, within the interaction interface that differs between PTPRM and PTPRK and show that mutation to glycine, the equivalent residue in PTPRM, abolishes PTPRK dimer formation in vitro. This comparison of two members of the receptor tyrosine phosphatase family suggests that homophilic specificity is driven by a combination of shape complementarity and specific but limited sequence differences.
|
Nov 2022
|
|
|
|
Jill
Trewhella
,
Patrice
Vachette
,
Jan
Bierma
,
Clement
Blanchet
,
Emre
Brookes
,
Srinivas
Chakravarthy
,
Leonie
Chatzimagas
,
Thomas E.
Cleveland
,
Nathan
Cowieson
,
Ben
Crossett
,
Anthony P.
Duff
,
Daniel
Franke
,
Frank
Gabel
,
Richard E.
Gillilan
,
Melissa
Graewert
,
Alexander
Grishaev
,
J. Mitchell
Guss
,
Michal
Hammel
,
Jesse
Hopkins
,
Qingqui
Huang
,
Jochen S.
Hub
,
Greg L.
Hura
,
Thomas C.
Irving
,
Cy Michael
Jeffries
,
Cheol
Jeong
,
Nigel
Kirby
,
Susan
Krueger
,
Anne
Martel
,
Tsutomu
Matsui
,
Na
Li
,
Javier
Pérez
,
Lionel
Porcar
,
Thierry
Prange
,
Ivan
Rajkovic
,
Mattia
Rocco
,
Daniel J.
Rosenberg
,
Timothy M.
Ryan
,
Soenke
Seifert
,
Hiroshi
Sekiguchi
,
Dmitri
Svergun
,
Susana
Teixeira
,
Aurelien
Thureau
,
Thomas M.
Weiss
,
Andrew E.
Whitten
,
Kathleen
Wood
,
Xiaobing
Zuo
Open Access
Abstract: Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å−1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å−1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å−1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.
|
Nov 2022
|
|
I03-Macromolecular Crystallography
|
Open Access
Abstract: Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.
|
Sep 2021
|
|
I03-Macromolecular Crystallography
|
Alex
Macpherson
,
Maisem
Laabei
,
Zainab
Ahdash
,
Melissa A
Graewert
,
James R
Birtley
,
Monika-Sarah
Schulze
,
Susan
Crennell
,
Sarah A
Robinson
,
Ben
Holmes
,
Vladas
Oleinikovas
,
Per H.
Nilsson
,
James
Snowden
,
Victoria
Ellis
,
Tom Eirik
Mollnes
,
Charlotte M.
Deane
,
Dmitri
Svergun
,
Alastair D. G.
Lawson
,
Jean M. H.
Van Den Elsen
Diamond Proposal Number(s):
[20029]
Open Access
Abstract: Bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce high affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.
|
Feb 2021
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Benjamin G.
Butt
,
Danielle J.
Owen
,
C. M.
Jeffries
,
Lyudmila
Ivanova
,
Chris H.
Hill
,
Jack W.
Houghton
,
Md Firoz
Ahmed
,
Robin
Antrobus
,
Dmitri I.
Svergun
,
John J.
Welch
,
Colin M.
Crump
,
Stephen C.
Graham
Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III–like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.
|
May 2020
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8659]
Open Access
Abstract: BACKGROUND: Casitas B-lineage lymphoma (Cbl or c-Cbl) is a RING ubiquitin ligase that negatively regulates protein tyrosine kinase (PTK) signalling. Phosphorylation of a conserved residue (Tyr371) on the linker helix region (LHR) between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available, they do not reveal how Tyr371 mutations affect Cbl's conformation. Here, we investigate how Tyr371 mutations affect Cbl's conformation in solution and how this relates to Cbl's ability to potentiate transformation in cells.
RESULTS: To explore how Tyr371 mutations affect Cbl's properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2-Ub), small angle X-ray scattering studies to investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation potential in cells. Cbl Tyr371 mutants enhance E2-Ub binding and cause Cbl to adopt extended conformations in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are those that perturb the LHR-substrate-binding domain interaction.
CONCLUSIONS: c-Cbl's LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl.
|
Sep 2016
|
|
B21-High Throughput SAXS
|
Abstract: The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.
|
Sep 2016
|
|
I04-Macromolecular Crystallography
|
David
Albesa-Jove
,
Natalia
Comino
,
Montse
Tersa
,
Elisabeth
Mohorko
,
Saioa
Urresti
,
Elisa
Dainese
,
Laurent R.
Chiarelli
,
Maria Rosalia
Pasca
,
Riccardo
Manganelli
,
Vadim
Makarov
,
Giovanna
Riccardi
,
Dmitri I.
Svergun
,
Rudi
Glockshuber
,
Marcelo
Guerin
Diamond Proposal Number(s):
[8302, 10130]
Open Access
Abstract: Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a ′closed′ compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an ′open′ conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.
|
Nov 2015
|
|
B21-High Throughput SAXS
|
Florian
Mittelberger
,
Cindy
Meyer
,
Georg H.
Waetzig
,
Martin
Zacharias
,
Erica
Valentini
,
Dmitri I.
Svergun
,
Katharina
Berg
,
Inken
Lorenzen
,
Joachim
Grötzinger
,
Stefan
Rose-John
,
Ulrich
Hahn
Abstract: Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule.
Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2′-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.
|
Jul 2015
|
|
I04-Macromolecular Crystallography
|
Dominika
Gruszka
,
Fiona
Whelan
,
Oliver E.
Farrance
,
Herman K. H.
Fung
,
Emanuele
Paci
,
Cy M.
Jeffries
,
Dmitri I.
Svergun
,
Clair
Baldock
,
Christoph G.
Baumann
,
David J.
Brockwell
,
Jennifer
Potts
,
Jane
Clarke
Diamond Proposal Number(s):
[7864]
Open Access
Abstract: Bacteria exploit surface proteins to adhere to other bacteria, surfaces and host cells. Such proteins need to project away from the bacterial surface and resist significant mechanical forces. SasG is a protein that forms extended fibrils on the surface of Staphylococcus aureus and promotes host adherence and biofilm formation. Here we show that although monomeric and lacking covalent cross-links, SasG maintains a highly extended conformation in solution. This extension is mediated through obligate folding cooperativity of the intrinsically disordered E domains that couple non-adjacent G5 domains thermodynamically, forming interfaces that are more stable than the domains themselves. Thus, counterintuitively, the elongation of the protein appears to be dependent on the inherent instability of its domains. The remarkable mechanical strength of SasG arises from tandemly arrayed ‘clamp’ motifs within the folded domains. Our findings reveal an elegant minimal solution for the assembly of monomeric mechano-resistant tethers of variable length.
|
Jun 2015
|
|
