scholarly journals Multi-state structure determination and dynamics analysis reveals a new ubiquitin-recognition mechanism in yeast ubiquitin C-terminal hydrolase

2021 ◽  
Author(s):  
Mayu Okada ◽  
Yutaka Tateishi ◽  
Eri Nojiri ◽  
Tsutomu Mikawa ◽  
Sundaresan Rajesh ◽  
...  
Keyword(s):  
Structure Determination ◽  
Active Site ◽  
Residual Dipolar Couplings ◽  
Biological Activities ◽  
Protein Structure Determination ◽  
Paramagnetic Nmr ◽  
Structural Basis ◽  
Recognition Mechanism ◽  
N Terminus ◽  
Large Motion

Despite accumulating evidence that protein dynamics is indispensable for understanding the structural basis of biological activities, it remains challenging to visualize the spatial description of the dynamics and to associate transient conformations with their molecular functions. We have developed a new NMR protein structure determination method for the inference of multi-state conformations using multiple types of NMR data, including paramagnetic NMR and residual dipolar couplings, as well as conventional NOEs. Integration of these data in the structure calculation permits delineating accurate ensemble structures of biomacromolecules. Applying the method to the protein yeast ubiquitin hydrolase 1 (YUH1), we find large dynamics of its N-terminus and crossover loop surrounding the active site for ubiquitin-recognition and proteolysis. The N-terminus gets into and out of the crossover loop, suggesting their underlying functional significance. Our results, including those from biochemical analysis, show that large motion surrounding the active site contributes strongly to the efficiency of the enzymatic activity.

scholarly journals An Integrative Approach to Determine 3D Protein Structures Using Sparse Paramagnetic NMR Data and Physical Modeling

2021 ◽  
Vol 8 ◽  
Author(s):  
Kari Gaalswyk ◽  
Zhihong Liu ◽  
Hans J. Vogel ◽  
Justin L. MacCallum
Keyword(s):  
Structure Determination ◽  
Residual Dipolar Couplings ◽  
Chemical Shifts ◽  
Protein Structures ◽  
Physical Modelling ◽  
Paramagnetic Nmr ◽  
Integrative Approach ◽  
Nmr Methods ◽  
Muscle Myosin

Paramagnetic nuclear magnetic resonance (NMR) methods have emerged as powerful tools for structure determination of large, sparsely protonated proteins. However traditional applications face several challenges, including a need for large datasets to offset the sparsity of restraints, the difficulty in accounting for the conformational heterogeneity of the spin-label, and noisy experimental data. Here we propose an integrative approach to structure determination combining sparse paramagnetic NMR with physical modelling to infer approximate protein structural ensembles. We use calmodulin in complex with the smooth muscle myosin light chain kinase peptide as a model system. Despite acquiring data from samples labeled only at the backbone amide positions, we are able to produce an ensemble with an average RMSD of ∼2.8 Å from a reference X-ray crystal structure. Our approach requires only backbone chemical shifts and measurements of the paramagnetic relaxation enhancement and residual dipolar couplings that can be obtained from sparsely labeled samples.

scholarly journals Tandem domain structure determination based on a systematic enumeration of conformations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thérèse E. Malliavin
Keyword(s):  
Structure Determination ◽  
Residual Dipolar Couplings ◽  
Distance Geometry ◽  
Protein Structure Determination ◽  
Conformational Space ◽  
Convergence Criterion ◽  
Saxs Data ◽  
Paramagnetic Resonance ◽  
Systematic Enumeration ◽  
Set Up

AbstractProtein structure determination is undergoing a change of perspective due to the larger importance taken in biology by the disordered regions of biomolecules. In such cases, the convergence criterion is more difficult to set up and the size of the conformational space is a obstacle to exhaustive exploration. A pipeline is proposed here to exhaustively sample protein conformations using backbone angle limits obtained by nuclear magnetic resonance (NMR), and then to determine the populations of conformations. The pipeline is applied to a tandem domain of the protein whirlin. An original approach, derived from a reformulation of the Distance Geometry Problem is used to enumerate the conformations of the linker connecting the two domains. Specifically designed procedure then permit to assemble the domains to the linker conformations and to optimize the tandem domain conformations with respect to two sets of NMR measurements: residual dipolar couplings and paramagnetic resonance enhancements. The relative populations of optimized conformations are finally determined by fitting small angle X-ray scattering (SAXS) data. The most populated conformation of the tandem domain is a semi-closed one, fully closed and more extended conformations being in minority, in agreement with previous observations. The SAXS and NMR data show different influences on the determination of populations.

scholarly journals Structural basis of pharmacological chaperoning for human β-galactosidase

2014 ◽  
Vol 70 (a1) ◽  
pp. C843-C843
Author(s):  
Hironori Suzuki ◽  
Umeharu Ohto ◽  
Katsumi Higaki ◽  
Teresa Mena-Barragán ◽  
Matilde Aguilar-Moncayo ◽  
...  
Keyword(s):  
Active Site ◽  
Neurodegenerative Disorder ◽  
Lysosomal Storage Diseases ◽  
Japanese Patients ◽  
Structural Basis ◽  
Lysosomal Storage ◽  
Active Site Residues ◽  
Recognition Mechanism ◽  
Gm1 Gangliosidosis ◽  
Pharmacological Chaperone

GM1-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-Galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation (ERAD) due to mutations in the β-Gal gene. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding, prevent ERAD and promote trafficking to the lysosome. Here, we present the enzymological properties of wild-type human β-Gal and two representative mutations in GM1 gangliosidosis Japanese patients (R201C and I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we determined the crystal structures of β-Gal in complex with these compouds and two structurally related analogues to elucidate the detailed atomic view of the recognition mechanism. All compounds bind to the active site of β-Gal with the sugar moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity and the PC potential are strongly affected by the mono or bicyclic structure of the core as well as the orientation, the nature and the length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.

scholarly journals Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Zhu Liu ◽  
Zhou Gong ◽  
Wen-Xue Jiang ◽  
Ju Yang ◽  
Wen-Kai Zhu ◽  
...  
Keyword(s):  
Target Recognition ◽  
Conformational Space ◽  
Paramagnetic Nmr ◽  
Structural Basis ◽  
Binding Affinities ◽  
Recognition Mechanism ◽  
Ubiquitin Chain ◽  
Conformational Selection ◽  
Nmr Data ◽  
Covalently Linked

A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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