Binding properties of the quaternary assembly protein SPAG1

2019 ◽  
Vol 476 (11) ◽  
pp. 1679-1694 ◽  
Author(s):  
Marie-Eve Chagot ◽  
Raphael Dos Santos Morais ◽  
Sana Dermouche ◽  
Dorian Lefebvre ◽  
Xavier Manival ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
3D Structure ◽  
Md Simulations ◽  
Specific Protein ◽  
Binding Interface ◽  
Motile Cells ◽  
Docking Approach ◽  
Shock Proteins ◽  
Quaternary Assembly

Abstract In cells, many constituents are able to assemble resulting in large macromolecular machineries possessing very specific biological and physiological functions, e.g. ribosome, spliceosome and proteasome. Assembly of such entities is commonly mediated by transient protein factors. SPAG1 is a multidomain protein, known to participate in the assembly of both the inner and outer dynein arms. These arms are required for the function of sensitive and motile cells. Together with RUVBL1, RUVBL2 and PIH1D2, SPAG1 is a key element of R2SP, a protein complex assisting the quaternary assembly of specific protein clients in a tissue-specific manner and associating with heat shock proteins (HSPs) and regulators. In this study, we have investigated the role of TPR domains of SPAG1 in the recruitment of HSP chaperones by combining biochemical assays, ITC, NMR spectroscopy and molecular dynamics (MD) simulations. First, we propose that only two, out of the three TPR domains, are able to recruit the protein chaperones HSP70 and HSP90. We then focused on one of these TPR domains and elucidated its 3D structure using NMR spectroscopy. Relying on an NMR-driven docking approach and MD simulations, we deciphered its binding interface with the C-terminal tails of both HSP70 and HSP90. Finally, we addressed the biological function of SPAG1 and specifically demonstrated that a SPAG1 sub-fragment, containing a putative P-loop motif, cannot efficiently bind and hydrolyze GTP in vitro. Our data challenge the interpretation of SPAG1 possessing GTPase activity. We propose instead that SPAG1 regulates nucleotide hydrolysis activity of the HSP and RUVBL1/2 partners.

Investigation of the structure of regulatory proteins interacting with glycosaminoglycans by combining NMR spectroscopy and molecular modeling – the beginning of a wonderful friendship

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Georg Künze ◽  
Daniel Huster ◽  
Sergey A. Samsonov
Keyword(s):  
Nmr Spectroscopy ◽  
Structural Information ◽  
Md Simulations ◽  
Specific Protein ◽  
Regulatory Proteins ◽  
Structural Constraints ◽  
Modeling Tools ◽  
Nuclear Overhauser ◽  
Nmr Methods ◽  
High Flexibility

Abstract The interaction of regulatory proteins with extracellular matrix or cell surface-anchored glycosaminoglycans (GAGs) plays important roles in molecular recognition, wound healing, growth, inflammation and many other processes. In spite of their high biological relevance, protein-GAG complexes are significantly underrepresented in structural databases because standard tools for structure determination experience difficulties in studying these complexes. Co-crystallization with subsequent X-ray analysis is hampered by the high flexibility of GAGs. NMR spectroscopy experiences difficulties related to the periodic nature of the GAGs and the sparse proton network between protein and GAG with distances that typically exceed the detection limit of nuclear Overhauser enhancement spectroscopy. In contrast, computer modeling tools have advanced over the last years delivering specific protein-GAG docking approaches successfully complemented with molecular dynamics (MD)-based analysis. Especially the combination of NMR spectroscopy in solution providing sparse structural constraints with molecular docking and MD simulations represents a useful synergy of forces to describe the structure of protein-GAG complexes. Here we review recent methodological progress in this field and bring up examples where the combination of new NMR methods along with cutting-edge modeling has yielded detailed structural information on complexes of highly relevant cytokines with GAGs.

scholarly journals The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6120
Author(s):  
Danielle M. Williams ◽  
David C. Thorn ◽  
Christopher M. Dobson ◽  
Sarah Meehan ◽  
Sophie E. Jackson ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Molecular Chaperones ◽  
Protein Unfolding ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Parkinson’S Diseases ◽  
Amino Acid Form ◽  
Shock Proteins

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Solution structure of the autophagy-related protein LC3C reveals a polyproline II motif on a mobile tether with phosphorylation site

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carsten Krichel ◽  
Christina Möckel ◽  
Oliver Schillinger ◽  
Pitter F. Huesgen ◽  
Heinrich Sticht ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Solution Structure ◽  
Phosphorylation Site ◽  
Backbone Dynamics ◽  
Dimensional Structure ◽  
High Sequence Identity ◽  
Amino Terminal ◽  
Polyproline Ii ◽  
Binding Interface

Abstract (Macro-)autophagy is a compartmental degradation pathway conserved from yeast to mammals. The yeast protein Atg8 mediates membrane tethering/hemifusion and cargo recruitment and is essential for autophagy. The human MAP1LC3/GABARAP family proteins show high sequence identity with Atg8, but MAP1LC3C is distinguished by a conspicuous amino-terminal extension with unknown functional significance. We have determined the high-resolution three-dimensional structure and measured the backbone dynamics of MAP1LC3C by NMR spectroscopy. From Ser18 to Ala120, MAP1LC3C forms an α-helix followed by the ubiquitin-like tertiary fold with two hydrophobic binding pockets used by MAP1LC3/GABARAP proteins to recognize targets presenting LC3-interacting regions (LIRs). Unlike other MAP1LC3/GABARAP proteins, the amino-terminal region of MAP1LC3C does not form a stable helix α1 but a “sticky arm” consisting of a polyproline II motif on a flexible linker. Ser18 at the interface between this linker and the structural core can be phosphorylated in vitro by protein kinase A, which causes additional conformational heterogeneity as monitored by NMR spectroscopy and molecular dynamics simulations, including changes in the LIR-binding interface. Based on these results we propose that the amino-terminal polyproline II motif mediates specific interactions with the microtubule cytoskeleton and that Ser18 phosphorylation modulates the interplay of MAP1LC3C with its various target proteins.

scholarly journals Combining SARS-CoV-2 Proofreading Exonuclease and RNA-Dependent RNA Polymerase Inhibitors as a Strategy to Combat COVID-19: A High-Throughput in silico Screening

2021 ◽  
Vol 12 ◽  
Author(s):  
Shradha Khater ◽  
Pawan Kumar ◽  
Nandini Dasgupta ◽  
Gautam Das ◽  
Shashikant Ray ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Free Energy ◽  
Solvent Accessibility ◽  
Nucleoside Analogs ◽  
Md Simulations ◽  
Rna Dependent Rna Polymerase ◽  
Born Model ◽  
Docking Approach ◽  
Approved Drugs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide. Currently, many clinical trials in search of effective COVID-19 drugs are underway. Viral RNA-dependent RNA polymerase (RdRp) remains the target of choice for prophylactic or curative treatment of COVID-19. Nucleoside analogs are the most promising RdRp inhibitors and have shown effectiveness in vitro, as well as in clinical settings. One limitation of such RdRp inhibitors is the removal of incorporated nucleoside analogs by SARS-CoV-2 exonuclease (ExoN). Thus, ExoN proofreading activity accomplishes resistance to many of the RdRp inhibitors. We hypothesize that in the absence of highly efficient antivirals to treat COVID-19, combinatorial drug therapy with RdRp and ExoN inhibitors will be a promising strategy to combat the disease. To repurpose drugs for COVID-19 treatment, 10,397 conformers of 2,240 approved drugs were screened against the ExoN domain of nsp14 using AutoDock VINA. The molecular docking approach and detailed study of interactions helped us to identify dexamethasone metasulfobenzoate, conivaptan, hesperidin, and glycyrrhizic acid as potential inhibitors of ExoN activity. The results were further confirmed using molecular dynamics (MD) simulations and molecular mechanics combined with generalized Born model and solvent accessibility method (MM-GBSA) calculations. Furthermore, the binding free energy of conivaptan and hesperidin, estimated using MM-GBSA, was −85.86 ± 0.68 and 119.07 ± 0.69 kcal/mol, respectively. Based on docking, MD simulations and known antiviral activities, and conivaptan and hesperidin were identified as potential SARS-CoV-2 ExoN inhibitors. We recommend further investigation of this combinational therapy using RdRp inhibitors with a repurposed ExoN inhibitor as a potential COVID-19 treatment.

scholarly journals Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

2018 ◽  
Vol 115 (17) ◽  
pp. E4071-E4080 ◽  
Author(s):  
Navneet Bung ◽  
Arijit Roy ◽  
Brenden Chen ◽  
Dibyajyoti Das ◽  
Meenakshi Pradhan ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Active Site ◽  
3D Structure ◽  
Acute Intermittent Porphyria ◽  
Md Simulations ◽  
Chain Elongation ◽  
Active Site Residues ◽  
Autosomal Dominant Disorder ◽  
Random Acceleration

Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human HMBS (hHMBS) cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme’s domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

Molecular Docking Approaches to Suggest the Anti-Mycobacterial Targets of Natural Products

2020 ◽  
Author(s):  
Rafael Baptista ◽  
Sumana Bhowmick ◽  
Shen Jianying ◽  
Luis Mur
Keyword(s):  
Natural Products ◽  
Molecular Docking ◽  
Free Energies ◽  
Antibiotic Resistant ◽  
Drug Lead ◽  
Bisbenzylisoquinoline Alkaloids ◽  
Docking Approach ◽  
Global Threat ◽  
Physicochemical And Structural Properties

Tuberculosis (TB) is a major global threat mostly due to the development of antibiotic resistant forms of Mycobacterium tuberculosis, the causal agent of the disease. Driven by the pressing need for new anti-mycobacterial agents, several natural products (NPs) have been shown to have in vitro activities against M. tuberculosis. The utility of any NP as a drug lead is augmented when the anti-mycobacterial target(s) is unknown. To suggest these, we used a molecular docking approach to predict the interactions of 53 selected anti-mycobacterial NPs against known ‘druggable’ mycobacterial targets ClpP1P2, DprE1, InhA, KasA, PanK, PknB and Pks13. The docking scores / binding free energies were predicted and calculated using AutoDock Vina along with physicochemical and structural properties of the NPs, using PaDEL descriptors. These were compared to the established inhibitor (control) drugs for each mycobacterial target. The specific interactions of the bisbenzylisoquinoline alkaloids 2-nortiliacorinine, tiliacorine and 13’-bromotiliacorinine against the targets PknB and DprE1 (-11.4, -10.9 and -9.8 kcal.mol-1 ; -12.7, -10.9 and -10.3 kcal.mol-1 , respectively) and the lignan αcubebin and Pks13 (-11.0 kcal.mol-1 ) had significantly superior docking scores compared to controls. Our approach can be used to suggest predicted targets for the NP to be validated experimentally but these in silico steps are likely to facilitate drug optimisation.

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