scholarly journals Conformational Changes in a Macrolide Antibiotic Binding Protein From Mycobacterium smegmatis Upon ADP Binding

2021 ◽  
Vol 12 ◽  
Author(s):  
Qingqing Zhang ◽  
Xiang Liu ◽  
Huijuan Liu ◽  
Bingjie Zhang ◽  
Haitao Yang ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Macrolide Antibiotic ◽  
Complex Structure ◽  
Binding Pocket ◽  
Similar Function ◽  
Transitional State ◽  
Abc Protein ◽  
Accessory Domain ◽  
Similar Conformation

Rv3197 (MABP-1), a non-canonical ABC protein in Mycobacterium tuberculosis, has ATPase activity and confers inducible resistance to the macrolide family of antibiotics. Here we have shown that MSMEG_1954, the homolog of Rv3197 in M. smegmatis, has a similar function of conferring macrolide resistance. Crystal structures of apo-MSMEG_1954 (form1 and form 2) and MSMEG_1954 in complex with ADP have been determined. These three structures show that MSMEG_1954 has at least two different conformations we identify as closed state (MSMEG_1954-form 1) and open state (MSMEG_1954-form 2 and MSMEG_1954-ADP). Structural superimposition shows that the MSMEG_1954-form 2 and MSMEG_1954-ADP complex have similar conformation to that observed for MABP-1 and MABP-1-erythromicin complex structure. However, the antibiotic binding pocket in MSMEG_1954-form 1 is completely blocked by the N-terminal accessory domain. When bound by ADP, the N-terminal accessory domain undergoes conformational change, which results in the open of the antibiotic binding pocket. Because of the degradation of N terminal accessory domain in MSMSG_1954-form 2, it is likely to represent a transitional state between MSMEG_1954-form 1 and MSMEG_1954-ADP complex structure.

Structural analysis of the Sil1–Bip complex reveals the mechanism for Sil1 to function as a nucleotide-exchange factor

2011 ◽  
Vol 438 (3) ◽  
pp. 447-455 ◽  
Author(s):  
Ming Yan ◽  
Jingzhi Li ◽  
Bingdong Sha
Keyword(s):  
Conformational Changes ◽  
Complex Structure ◽  
Binding Pocket ◽  
Atpase Domain ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Complex Crystal Structure ◽  
Adp Release ◽  
Complex Crystal

Sil1 functions as a NEF (nucleotide-exchange factor) for the ER (endoplasmic reticulum) Hsp70 (heat-shock protein of 70 kDa) Bip in eukaryotic cells. Sil1 may catalyse the ADP release from Bip by interacting directly with the ATPase domain of Bip. In the present study we show the complex crystal structure of the yeast Bip and the NEF Sil1 at the resolution of 2.3 Å (1 Å=0.1 nm). In the Sil1–Bip complex structure, the Sil1 molecule acts as a ‘clamp’ which binds lobe IIb of the Bip ATPase domain. The binding of Sil1 causes the rotation of lobe IIb ~ 13.5° away from the ADP-binding pocket. The complex formation also induces lobe Ib to swing in the opposite direction by ~ 3.7°. These conformational changes open up the nucleotide-binding pocket in the Bip ATPase domain and disrupt the hydrogen bonds between Bip and bound ADP, which may catalyse ADP release. Mutation of the Sil1 residues involved in binding the Bip ATPase domain compromise the binding affinity of Sil1 to Bip, and these Sil1 mutants also abolish the ability to stimulate the ATPase activity of Bip.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

scholarly journals How to build a water-splitting machine: structural insights into photosystem II assembly

2020 ◽  
Author(s):  
Jure Zabret ◽  
Stefan Bohn ◽  
Sandra Schuller ◽  
Oliver Arnolds ◽  
Madeline Möller ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Water Splitting ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Heme Iron ◽  
Structural Motif ◽  
Acceptor Side ◽  
Non Heme Iron ◽  
Assembly Intermediate ◽  
Stepwise Assembly

Abstract Biogenesis of photosystem II (PSII), nature’s water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate with 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace bicarbonate with glutamate as a ligand of the non-heme iron, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction.

scholarly journals Streptavidin/biotin: Tethering geometry defines unbinding mechanics

2020 ◽  
Vol 6 (13) ◽  
pp. eaay5999 ◽  
Author(s):  
Steffen M. Sedlak ◽  
Leonard C. Schendel ◽  
Hermann E. Gaub ◽  
Rafael C. Bernardi
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Energy Barrier ◽  
Mechanical Stability ◽  
Binding Pocket ◽  
Entire System ◽  
Shear Forces ◽  
Single Molecule Force Spectroscopy ◽  
Force Loading ◽  
Applied Forces

Macromolecules tend to respond to applied forces in many different ways. Chemistry at high shear forces can be intriguing, with relatively soft bonds becoming very stiff in specific force-loading geometries. Largely used in bionanotechnology, an important case is the streptavidin (SA)/biotin interaction. Although SA’s four subunits have the same affinity, we find that the forces required to break the SA/biotin bond depend strongly on the attachment geometry. With AFM-based single-molecule force spectroscopy (SMFS), we measured unbinding forces of biotin from different SA subunits to range from 100 to more than 400 pN. Using a wide-sampling approach, we carried out hundreds of all-atom steered molecular dynamics (SMD) simulations for the entire system, including molecular linkers. Our strategy revealed the molecular mechanism that causes a fourfold difference in mechanical stability: Certain force-loading geometries induce conformational changes in SA’s binding pocket lowering the energy barrier, which biotin has to overcome to escape the pocket.

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

scholarly journals Comparative Analyses of theβ-Tubulin Gene and Molecular Modeling Reveal Molecular Insight into the Colchicine Resistance in Kinetoplastids Organisms

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Luis Luis ◽  
María Luisa Serrano ◽  
Mariana Hidalgo ◽  
Alexis Mendoza-León
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Differential Susceptibility ◽  
Binding Pocket ◽  
Crystallographic Structure ◽  
Tubulin Gene ◽  
Colchicine Binding Site ◽  
Colchicine Resistance ◽  
Leishmania Spp

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such asLeishmania spp. andTrypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site ofLeishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced theβ-tubulin gene ofLeishmania (Viannia) guyanensisand established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on theLeishmania  β-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in theβ-tubulin sequence of kinetoplastid organisms such asTrypanosoma cruzi,T. brucei, andT. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of anα-helix structure and displacement to the inside of the pocket of oneβ-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.

scholarly journals Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser

2019 ◽  
Vol 73 (8-9) ◽  
pp. 509-518
Author(s):  
Jienv Ding ◽  
Monalisa Swain ◽  
Ping Yu ◽  
Jason R. Stagno ◽  
Yun-Xing Wang
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Free Electron Laser ◽  
Messenger Rna ◽  
Bound States ◽  
Conformational Dynamics ◽  
Thermal Fluctuation ◽  
Binding Pocket ◽  
X Ray ◽  
Aptamer Domain

Abstract Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s−1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s−1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s−1. The µs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

scholarly journals GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

1993 ◽  
Vol 122 (6) ◽  
pp. 1253-1265 ◽  
Author(s):  
GW Kemble ◽  
YI Henis ◽  
JM White
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Binding Activity ◽  
Great Distance ◽  
Fusion Peptides ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Receptor Binding Activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.

Synthetic site-directed ligands

1989 ◽  
Vol 323 (1217) ◽  
pp. 495-509 ◽  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Opioid Peptides ◽  
Binding Pocket ◽  
Tyrosine Residues ◽  
Chemotactic Peptides ◽  
Binding Cavity ◽  
False Floor ◽  
Type Site ◽  
Main Cavity

Complexes of nucleotides, peptides and arom atic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. Alter tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04- 01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the ‘native’ protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Meg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the ε-DNP ring near the floor of the main cavity and the α-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with both DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Meg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.

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