I03-Macromolecular Crystallography
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Abstract: Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy—“mash-up” design—to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.
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Apr 2021
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I04-Macromolecular Crystallography
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Open Access
Abstract: The light chains (KLCs) of the heterotetrameric microtubule motor kinesin-1, that bind to cargo adaptor proteins and regulate its activity, have a capacity to recognize short peptides via their tetratricopeptide repeat domains (KLCTPR). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call ‘Y-acidic’ (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1TPR are distinct from those utilized for the recognition of W-acidic motifs found in adaptors that are KLC- isoform non-selective. However, a partial overlap on their receptor binding sites implies that adaptors relying on Y-acidic and W-acidic motifs must act independently. We propose a model to explain why these two classes of motifs that bind to the concave surface of KLCTPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity.
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Oct 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Open Access
Abstract: The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.
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Jan 2017
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I24-Microfocus Macromolecular Crystallography
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Abstract: The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLCTPR), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.
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Feb 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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C. X.
Santos
,
A. D.
Hafstad
,
M.
Beretta
,
M.
Zhang
,
C.
Molenaar
,
J.
Kopec
,
D.
Fotinou
,
T. V.
Murray
,
A. M.
Cobb
,
D.
Martin
,
M.
Zeh Silva
,
N.
Anilkumar
,
K.
Schroder
,
C. M.
Shanahan
,
A. C.
Brewer
,
R. P.
Brandes
,
E.
Blanc
,
M.
Parsons
,
V.
Belousov
,
R.
Cammack
,
R. C.
Hider
,
R. A.
Steiner
,
A. M.
Shah
Diamond Proposal Number(s):
[7656]
Open Access
Abstract: Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine–threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species‐generating NADPH oxidase‐4 (Nox4) is induced downstream of ATF4, binds to a PP1‐targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4‐regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia–reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4–GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.
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Jan 2016
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I02-Macromolecular Crystallography
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Abstract: Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.
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Dec 2014
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1220]
Abstract: HIV-1 and other enveloped viruses can be restricted by a host cellular protein called BST2/tetherin that prevents release of budded viruses from the cell surface. Mature BST2 contains a small cytosolic region, a predicted transmembrane helix, and an extracellular domain with a C-terminal GPI anchor. To advance understanding of BST2 function, we have determined a 2.6 Å crystal structure of the extracellular domain of the bacterially expressed recombinant human protein, residues 47–152, under reducing conditions. The structure forms a single long helix that associates as a parallel dimeric coiled coil over its C-terminal two-thirds, while the N-terminal third forms an antiparallel four-helix bundle with another dimer, creating a global tetramer. We also report the 3.45 Å resolution structure of BST2(51-151) prepared by expression as a secreted protein in HEK293T cells. This oxidized construct forms a dimer in the crystal that is superimposable with the reduced protein over the C-terminal two-thirds of the molecule, and its N terminus suggests pronounced flexibility. Hydrodynamic data demonstrated that BST2 formed a stable tetramer under reducing conditions and a dimer when oxidized to form disulfide bonds. A mutation that selectively disrupted the tetramer (L70D) increased protein expression modestly but only reduced antiviral activity by approximately threefold. Our data raise the possibility that BST2 may function as a tetramer at some stage, such as during trafficking, and strongly support a model in which the primary functional state of BST2 is a parallel disulfide-bound coiled coil that displays flexibility toward its N terminus.
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Oct 2010
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1220]
Abstract: Whereas the majority of O(2)-metabolizing enzymes depend on transition metal ions or organic cofactors for catalysis, a significant number of oxygenases and oxidases neither contain nor require any cofactor. Among the cofactor-independent oxidases, urate oxidase, coproporphyrinogen oxidase, and formylglycine-generating enzyme are of mechanistic as well as medical interest. Formylglycine-generating enzyme is also a promising tool for protein engineering as it can be used to equip proteins with a reactive aldehyde function. PqqC, an oxidase in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, catalyzes an eight-electron ring-closure oxidation reaction. Among bacterial oxygenases, quinone-forming monooxygenases involved in the tailoring of polyketides, the dioxygenase DpgC found in the biosynthesis of a building block of vancomycin and teicoplanin antibiotics, luciferase monooxygenase from Renilla sp., and bacterial ring-cleaving 2,4-dioxygenases active towards 3-hydroxy-4(1H)-quinolones have been identified as cofactor-independent enzymes. Interestingly, the 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases as well as Renilla luciferase use an alpha/beta-hydrolase architecture for oxygenation reactions. Cofactor-independent oxygenases and oxidases catalyze very different reactions and belong to several different protein families, reflecting their diverse origin. Nevertheless, they all may share the common mechanistic concept of initial base-catalyzed activation of their organic substrate and "substrate-assisted catalysis
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Apr 2010
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1220]
Abstract: Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Rü61a and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the ?/?-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1-H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical ?/?-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The “oxyanion hole” of the ?/?-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions.
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Jan 2010
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1220]
Abstract: 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) is a cofactor-devoid dioxygenase that is involved in the anthranilate pathway of quinaldine degradation. HOD has been proposed to belong to the [alpha]/[beta]-hydrolase-fold superfamily of enzymes. N-terminally His6-tagged HOD has been crystallized by the hanging-drop vapour-diffusion method using sodium/potassium tartrate as a precipitant and CuCl2 as an additive. The structure was solved by the single anomalous dispersion (SAD) technique using data collected to 3.5 Å resolution at the Cu absorption peak wavelength. The crystals belong to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 153.788, c = 120.872 Å.
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May 2007
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