scholarly journals A Ubiquitin-Binding Domain that Does Not Bind Ubiquitin

2018 ◽  
Author(s):  
Michael Lim ◽  
Joseph A. Newman ◽  
Hannah L. Williams ◽  
Hazel Aitkenhead ◽  
Opher Gileadi ◽  
...  
Keyword(s):  
Structural Model ◽  
Coiled Coil ◽  
Biological Processes ◽  
Hydrophobic Residues ◽  
X Ray ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Ubiquitin Binding ◽  
Ubiquitin Binding Domain ◽  
Cue Domain

Ubiquitylation, the post-translational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function exclusively by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. We show that the SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. A structural model of the minimal KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography analysis is presented. Remarkably, the CUE1 domain, which resembles a canonical CUE domain, recognizes 2 clusters of exposed hydrophobic residues on KAP1, but these are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data challenge the well-established dogma that CUE domains exclusively recognize the ubiquitin-fold.

scholarly journals CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 284 ◽  
Author(s):  
Ridvan Nepravishta ◽  
Federica Ferrentino ◽  
Walter Mandaliti ◽  
Anna Mattioni ◽  
Janine Weber ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Functional Characterization ◽  
Binding Domain ◽  
15N Nmr ◽  
Protein Sequence Analysis ◽  
Hydrophobic Patch ◽  
Binding Domains ◽  
Ubiquitin Binding ◽  
Ubiquitin Binding Domain

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

scholarly journals Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

2018 ◽  
Author(s):  
Andrew Lyon ◽  
Alex Zelter ◽  
Shruthi Viswanath ◽  
Alison Maxwell ◽  
Richard Johnson ◽  
...  
Keyword(s):  
Structural Model ◽  
Coiled Coil ◽  
The Other ◽  
Assembly Process ◽  
X Ray ◽  
X Ray Crystallography ◽  
Small Complex ◽  
Ring Complex ◽  
Biochemical Analyses

AbstractMicrotubule (MT) nucleation in vivo is regulated by the γ-tubulin ring complex (γTuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast γTuRC is composed of seven γ-tubulin small complex (γTuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple γTuSCs, thereby stabilizing the otherwise low-affinity interface between γTuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with γTuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the γTuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and γTuSC and the other stabilizing the interface between adjacent γTuSCs.

scholarly journals Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

2010 ◽  
Vol 84 (9) ◽  
pp. 4706-4713 ◽  
Author(s):  
François Hoh ◽  
Marilyne Uzest ◽  
Martin Drucker ◽  
Célia Plisson-Chastang ◽  
Patrick Bron ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Coiled Coil ◽  
Virus Transmission ◽  
Cauliflower Mosaic Virus ◽  
Insect Vector ◽  
Virus Surface ◽  
X Ray Crystallography ◽  
Coil Structure

ABSTRACT Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.

scholarly journals Structure and mechanism of ATP-binding cassette transporters

2008 ◽  
Vol 364 (1514) ◽  
pp. 239-245 ◽  
Author(s):  
Kaspar P Locher
Keyword(s):  
Abc Transporters ◽  
Integral Membrane Proteins ◽  
Atp Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Mechanistic Insight ◽  
Future Challenges ◽  
Hydrolysis Of ◽  
Atp Binding Cassette

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined.

scholarly journals Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Sebastian H.W. Kraatz ◽  
Sarah Bianchi ◽  
Michel O. Steinmetz
Keyword(s):  
Coiled Coil ◽  
Coiled Coils ◽  
Molecular Replacement ◽  
Disulfide Bridges ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Eukaryotic Proteins ◽  
Sad Phasing

Coiled-coils are ubiquitous protein–protein interaction motifs found in many eukaryotic proteins. The elongated, flexible and often irregular nature of coiled-coils together with their tendency to form fibrous arrangements in crystals imposes challenges on solving the phase problem by molecular replacement. Here, we report the successful combinatorial use of native and rational engineered disulfide bridges together with sulfur-SAD phasing as a powerful tool to stabilize and solve the structure of coiled-coil domains in a straightforward manner. Our study is a key example of how modern sulfur SAD combined with mutagenesis can help to advance and simplify the structural study of challenging coiled-coil domains by X-ray crystallography.

scholarly journals Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2109-2117 ◽  
Author(s):  
Meghan A. Rego ◽  
Frederick W. Kolling ◽  
Elizabeth A. Vuono ◽  
Maurizio Mauro ◽  
Niall G. Howlett
Keyword(s):  
Fanconi Anemia ◽  
Noncovalent Interaction ◽  
Binding Domain ◽  
Covalent Attachment ◽  
Pathway Activation ◽  
Ubiquitin Binding ◽  
Ubiquitin Binding Domain ◽  
Ubiquitin Conjugation ◽  
Cue Domain

Abstract The Fanconi anemia (FA)–BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that although mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2, and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation.

scholarly journals Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

2005 ◽  
Vol 390 (1) ◽  
pp. 115-123 ◽  
Author(s):  
James Sandy ◽  
Adeel Mushtaq ◽  
Simon J. Holton ◽  
Pamela Schartau ◽  
Martin E. M. Noble ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Structural Model ◽  
Sequence Data ◽  
Structural Features ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Acetyl Transfer ◽  
Insight Into

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

Solution and solid-state characterization of a dicopper receptor for large substrates

2002 ◽  
Vol 80 (5) ◽  
pp. 496-498 ◽  
Author(s):  
Greg JE Davidson ◽  
Andrew J Baer ◽  
Adrien P Côté ◽  
Nicholas J Taylor ◽  
Garry S Hanan ◽  
...  
Keyword(s):  
Metal Binding ◽  
Central Cavity ◽  
X Ray ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Rigid Spacer ◽  
Complex Coordination ◽  
Solid State Characterization ◽  
Large Central Cavity

Ligand 2 contains two metal-binding domains separated by a rigid spacer and assembles into a dicopper receptor with a large central cavity with no evidence of catenation.Key words: copper complex, coordination, X-ray crystallography, molecular recognition.

scholarly journals An anticipated optimization approach to macromolecular crystallization

2019 ◽  
Author(s):  
Fabrice Gorrec
Keyword(s):  
Cost Effective ◽  
Optimization Approach ◽  
Biological Processes ◽  
Initial Sample ◽  
X Ray ◽  
X Ray Crystallography ◽  
Starting Point ◽  
Macromolecular Crystallization ◽  
As Solubility

AbstractCrystallization is an essential step for determining macromolecular structures at atomic resolution with X-ray crystallography. Crystals of diffraction quality obtained from purified samples of proteins, RNAs, DNAs and their complexes enable our understanding of biological processes and structure-based design of drugs. Targets of interest for researchers are however increasingly challenging to produce and crystallise. Progress in crystallization methods applicable to limiting amount of sample while increasing the yield of useful crystals are hence urgently needed. In this context, an Anticipated Optimization Approach was investigated. For this approach, it is assumed that samples are highly unstable and will most probably not produce useful crystals (‘hits’). By selecting leads very early, what remains from the initial sample can be used for follow-up optimization experiments. Subsequently, the reproducibility issues caused by sample variability are bypassed. An initial crystallization screen that failed to produce hits becomes a well-suited solubility assay that is a starting point for optimization. The approach was tested with a straightforward and cost-effective protocol developed elsewhere. The yield of useful crystals was increased and accelerated for three targets of pharmacologic studies.SynopsisThis study suggests that the yield of useful crystals obtained from challenging protein samples can be increased by using an initial crystallization screen as solubility assay.

scholarly journals The Diamond Light Source and the challenges ahead for structural biology: some informal remarks

2015 ◽  
Vol 373 (2036) ◽  
pp. 20130156 ◽  
Author(s):  
V. Ramakrishnan
Keyword(s):  
Synchrotron Radiation ◽  
Light Source ◽  
Structural Biology ◽  
Complex Structures ◽  
Biological Processes ◽  
Short Article ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past

The remarkable advances in structural biology in the past three decades have led to the determination of increasingly complex structures that lie at the heart of many important biological processes. Many of these advances have been made possible by the use of X-ray crystallography using synchrotron radiation. In this short article, some of the challenges and prospects that lie ahead will be summarized.

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