I04-1-Macromolecular Crystallography (fixed wavelength)
|
Sarah
Rollauer
,
Michael J.
Tarry
,
James E.
Graham
,
Mari
Jääskeläinen
,
Franziska
Jäger
,
Steven
Johnson
,
Martin
Krehenbrink
,
Sai-Man
Liu
,
Michael J.
Lukey
,
Julien
Marcoux
,
Melanie A.
Mcdowell
,
Fernanda
Rodriguez
,
Pietro
Roversi
,
Phillip J.
Stansfeld
,
Carol V.
Robinson
,
Mark S. P.
Sansom
,
Tracy
Palmer
,
Martin
Högbom
,
Ben C.
Berks
,
Susan M.
Lea
Abstract: The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.
|
Dec 2012
|
|
I24-Microfocus Macromolecular Crystallography
|
A.
Quigley
,
Y. Y.
Dong
,
A. C. W.
Pike
,
L.
Dong
,
L.
Shrestha
,
G.
Berridge
,
P. J.
Stansfeld
,
M. S. P.
Sansom
,
A. M.
Edwards
,
C.
Bountra
,
F.
Von Delft
,
A. N.
Bullock
,
N. A.
Burgess-Brown
,
E. P.
Carpenter
Diamond Proposal Number(s):
[8421]
Abstract: Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane ?-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide–binding site and to the bottom of the chamber.
|
Mar 2013
|
|
I24-Microfocus Macromolecular Crystallography
|
Chitra A.
Shintre
,
Ashley C. W.
Pike
,
Quihong
Li
,
Jung-In
Kim
,
Alistair J.
Barr
,
Solenne
Goubin
,
Leela
Shrestha
,
Jingjie
Yang
,
Georgina
Berridge
,
Jonathan
Ross
,
Phillip J.
Stansfeld
,
Mark S. P.
Sansom
,
Aled M.
Edwards
,
Chas
Bountra
,
Brian D.
Marsden
,
Frank
Von Delft
,
Alex N.
Bullock
,
Opher
Gileadi
,
Nicola A.
Burgess-Brown
,
Elisabeth P.
Carpenter
Open Access
Abstract: ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.
|
May 2013
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Open Access
Abstract: Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal “strut” that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections.
|
Nov 2013
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[7641]
Abstract: Lipopolysaccharide (LPS) is essential for most Gram-negative bacteria and has crucial roles in protection of the bacteria from harsh environments and toxic compounds, including antibiotics. Seven LPS transport proteins (that is, LptA–LptG) form a trans-envelope protein complex responsible for the transport of LPS from the inner membrane to the outer membrane, the mechanism for which is poorly understood. Here we report the first crystal structure of the unique integral membrane LPS translocon LptD–LptE complex. LptD forms a novel 26-stranded β-barrel, which is to our knowledge the largest β-barrel reported so far. LptE adopts a roll-like structure located inside the barrel of LptD to form an unprecedented two-protein ‘barrel and plug’ architecture. The structure, molecular dynamics simulations and functional assays suggest that the hydrophilic O-antigen and the core oligosaccharide of the LPS may pass through the barrel and the lipid A of the LPS may be inserted into the outer leaflet of the outer membrane through a lateral opening between strands β1 and β26 of LptD. These findings not only help us to understand important aspects of bacterial outer membrane biogenesis, but also have significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.
|
Jun 2014
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[9306]
Open Access
Abstract: TolR is a 15-kDa inner membrane protein subunit of the Tol-Pal complex in Gram- negative bacteria the function of which is poorly understood. Tol-Pal is recruited to cell division sites where it is involved in maintaining the integrity of the outer membrane. TolR is related to MotB, the peptidoglycan (PG)-binding stator protein from the flagellum, suggesting it might serve a similar role in Tol-Pal. The only structure thus far reported for TolR is of the periplasmic domain from Haemophilus influenzae in which N- and C-terminal residues had been deleted (TolR62-133, E. coli numbering). H. influenzae TolR62-133 is a symmetrical dimer with a large deep cleft at the dimer interface. Here, we present the 1.7 Å crystal structure of the intact periplasmic domain of Escherichia coli TolR (TolR36-142). E. coli TolR36-142 is also dimeric but the architecture of the dimer is radically different to that of TolR62-133 due to intertwining of its N- and C-termini. TolR monomers are rotated ~180 ° relative to each other as a result of this strand-swapping, obliterating the putative PG-binding groove seen in TolR62-133. We found that removal of the strand swapped regions (TolR60-133) exposes cryptic PG binding activity that is absent in the full-length domain. We conclude that to function as a stator in the Tol-Pal complex dimeric TolR must undergo large- scale structural remodelling reminiscent of that proposed for MotB where N- and C-terminal sequences unfold in order for the protein to both reach and bind the PG layer ~90 Å away from the inner membrane.
|
Sep 2015
|
|
I24-Microfocus Macromolecular Crystallography
|
Dianfan
Li
,
Phillip J.
Stansfeld
,
Mark S. P.
Sansom
,
Aaron
Keogh
,
Lutz
Vogeley
,
Nicole
Howe
,
Joseph
Lyons
,
David
Aragao
,
Petra
Fromme
,
Raimund
Fromme
,
Shibom
Basu
,
Ingo
Grotjohann
,
Christopher
Kupitz
,
Kimberley
Rendek
,
Uwe
Weierstall
,
Nadia A.
Zatsepin
,
Vadim
Cherezov
,
Wei
Liu
,
Sateesh
Bandaru
,
Niall J.
English
,
Cornelius
Gati
,
Anton
Barty
,
Oleksandr
Yefanov
,
Henry N.
Chapman
,
Kay
Diederichs
,
Marc
Messerschmidt
,
Sébastien
Boutet
,
Garth J.
Williams
,
M.
Marvin Seibert
,
Martin
Caffrey
Open Access
Abstract: Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
|
Dec 2015
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Yinghong
Gu
,
Huanyu
Li
,
Haohao
Dong
,
Yi
Zeng
,
Zhengyu
Zhang
,
Neil G.
Paterson
,
Phillip J.
Stansfeld
,
Zhongshan
Wang
,
Yizheng
Zhang
,
Wenjian
Wang
,
Changjiang
Dong
Diamond Proposal Number(s):
[9475]
Abstract: All Gram-negative bacteria, mitochondria and chloroplasts have outer membrane proteins (OMPs) that perform many fundamental biological processes. The OMPs in Gram-negative bacteria are inserted and folded into the outer membrane by the β-barrel assembly machinery (BAM). The mechanism involved is poorly understood, owing to the absence of a structure of the entire BAM complex. Here we report two crystal structures of the Escherichia coli BAM complex in two distinct states: an inward-open state and a lateral-open state. Our structures reveal that the five polypeptide transport-associated domains of BamA form a ring architecture with four associated lipoproteins, BamB–BamE, in the periplasm. Our structural, functional studies and molecular dynamics simulations indicate that these subunits rotate with respect to the integral membrane β-barrel of BamA to induce movement of the β-strands of the barrel and promote insertion of the nascent OMP.
|
Feb 2016
|
|
I24-Microfocus Macromolecular Crystallography
|
Abstract: With functions that range from cell envelope structure to signal transduction and transport, lipoproteins constitute 2 to 3% of bacterial genomes and play critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Lipoproteins are synthesized with a signal peptide securing them to the cytoplasmic membrane with the lipoprotein domain in the periplasm or outside the cell. Posttranslational processing requires a signal peptidase II (LspA) that removes the signal peptide. Here, we report the crystal structure of LspA from Pseudomonas aeruginosa complexed with the antimicrobial globomycin at 2.8 angstrom resolution. Mutagenesis studies identify LspA as an aspartyl peptidase. In an example of molecular mimicry, globomycin appears to inhibit by acting as a noncleavable peptide that sterically blocks the active site. This structure should inform rational antibiotic drug discovery.
|
Feb 2016
|
|
I24-Microfocus Macromolecular Crystallography
|
Maciej
Wiktor
,
Dietmar
Weichert
,
Nicole
Howe
,
Chia-Ying
Huang
,
Vincent
Olieric
,
Coilín
Boland
,
Jonathan
Bailey
,
Lutz
Vogeley
,
Phillip J.
Stansfeld
,
Nienke
Buddelmeijer
,
Meitian
Wang
,
Martin
Caffrey
Diamond Proposal Number(s):
[11890, 12710]
Open Access
Abstract: Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.
|
Jul 2017
|
|
