scholarly journals Structural basis of adenylyl cyclase 9 activation

2021 ◽  
Author(s):  
Chao Qi ◽  
Pia Lavriha ◽  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Inayathulla Mohammed ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Site ◽  
Adenylyl Cyclase ◽  
Conformational Changes ◽  
Structural Basis ◽  
Structural Studies ◽  
Nucleotide Analogue ◽  
C Terminus ◽  
Membrane Bound ◽  
A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

scholarly journals Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854→Leu)

1996 ◽  
Vol 317 (2) ◽  
pp. 509-514 ◽  
Author(s):  
James M. SALHANY ◽  
Renee L. SLOAN ◽  
Lawrence M. SCHOPFER
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Covalent Binding ◽  
Band 3 ◽  
Blood Group Antigen ◽  
Cross Linking ◽  
Anion Exchange Protein ◽  
Membrane Bound ◽  
And Control

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases

2018 ◽  
Vol 475 (6) ◽  
pp. 1141-1158 ◽  
Author(s):  
Erika Artukka ◽  
Heidi H. Luoto ◽  
Alexander A. Baykov ◽  
Reijo Lahti ◽  
Anssi M. Malinen
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Systematic Analysis ◽  
Change Mechanism ◽  
Excess Substrate ◽  
Na Ions ◽  
Membrane Bound ◽  
A Site ◽  
Pyrophosphate Hydrolysis

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H+ and/or Na+ ions, are divided into K+,Na+-independent, Na+-regulated, and K+-dependent families. The first two families include H+-transporting mPPases (H+-PPases), whereas the last family comprises one Na+-transporting, two Na+- and H+-transporting subfamilies (Na+-PPases and Na+,H+-PPases, respectively), and three H+-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K+ binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K+-independent mPPases. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4–7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

scholarly journals Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR

2018 ◽  
Vol 115 (36) ◽  
pp. 8978-8983 ◽  
Author(s):  
Caillan Crowe-McAuliffe ◽  
Michael Graf ◽  
Paul Huter ◽  
Hiraku Takada ◽  
Maha Abdelshahid ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Subtilis ◽  
Binding Site ◽  
Pathogenic Bacteria ◽  
Small Subunit ◽  
Structural Basis ◽  
Abc Protein ◽  
Peptidyltransferase Center ◽  
A Site ◽  
Microbiological Analyses

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).

scholarly journals Activation of the Redox-regulated Chaperone Hsp33 by Domain Unfolding

2004 ◽  
Vol 279 (19) ◽  
pp. 20529-20538 ◽  
Author(s):  
Paul C. F. Graf ◽  
Maria Martinez-Yamout ◽  
Stephen VanHaerents ◽  
Hauke Lilie ◽  
H. Jane Dyson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Binding Site ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Helical Structure ◽  
Activation Process ◽  
Spectroscopic Techniques ◽  
Substrate Binding Site ◽  
C Terminus ◽  
Dimerization Interface

The molecular chaperone Hsp33 inEscherichia coliresponds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, α-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.

Abstract 393: Covalently Connected Apolipoprotein A-I Molecules Provide Support for the Double Belt Model in Reconstituted HDL

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jamie C Morris ◽  
W. Sean Davidson
Keyword(s):  
Critical Role ◽  
Density Lipoprotein ◽  
Bound State ◽  
Sodium Cholate ◽  
Structural Basis ◽  
Structural Studies ◽  
Wild Type ◽  
Apolipoprotein A ◽  
C Terminus ◽  
Hdl Metabolism

Because of its critical role in high density lipoprotein (HDL) metabolism, significant work has been devoted to studying apolipoprotein (apo)A-I structure in its lipid-bound state, particularly reconstituted forms of HDL (rHDL). Unfortunately, high-resolution structural studies have not been successful because of HDL heterogeneity and compositional dynamics. There is general agreement that apoA-I adopts an antiparallel arrangement on rHDL with the majority of the molecule conforming to the “double belt” model. However, less is understood about the locations of the N- and C-termini in these particles. The classic double belt predicts that, in a particle containing two molecules of apoA-I, all four termini are localized to one area of the particle. However, other models predict that they are far apart. To address this issue, we generated a series of covalently connected apoA-I dimers and tested their ability to generate rHDL particles. The dimeric (d)-apoA-I C-N mutant, locked the C-terminus of one apoA-I molecule to the N-terminus of a second molecule with three intervening Ala residues. Linked by strategically introduced cysteine residues, the d-apoA-I C-C , and d-apoA-I N-N mutants lock the C-termini and the N-termini of two apoA-I molecules together, respectively. When all three of these mutants were reconstituted into rHDL particles using sodium cholate and synthetic phospholipids, the resulting particles closely resembled those generated with wild-type (WT) apoA-I. This indicates a ring-like structure predicted by the double belt. However, when testing the ability of these mutants to spontaneously clear lipid, we found that both the N- and C- termini required the ability to separate and move independently to form normal particles. Native gel analysis showed the dimerized lipid-free mutants present as a tight homogenous band compared to wild-type which runs as a mixture of oligomers. We are currently evaluating if the homogeneity of these mutants can allow for more facile crystallization of full-length apoA-I. Further studies with these stabilized mutants should help us understand the structural basis underlying HDL’s diverse functions, which is essential for developing HDL-based therapies for cardiovascular disease.

scholarly journals Mechanism of tRNA-mediated +1 ribosomal frameshifting

2018 ◽  
Vol 115 (44) ◽  
pp. 11226-11231 ◽  
Author(s):  
Samuel Hong ◽  
S. Sunita ◽  
Tatsuya Maehigashi ◽  
Eric D. Hoffer ◽  
Jack A. Dunkle ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Elongation Factor ◽  
Amino Acid Sequences ◽  
Structural Basis ◽  
Forward Movement ◽  
Stem Loop ◽  
Bacterial Ribosome ◽  
Conformational Rearrangements ◽  
A Site

Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA–mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

scholarly journals Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

2007 ◽  
Vol 130 (2) ◽  
pp. 157-168 ◽  
Author(s):  
David M. Blodgett ◽  
Julie K. De Zutter ◽  
Kara B. Levine ◽  
Pusha Karim ◽  
Anthony Carruthers
Keyword(s):  
Glucose Transport ◽  
Conformational Changes ◽  
Covalent Modification ◽  
Antibody Binding ◽  
Mass Spectrometry Analysis ◽  
Structural Basis ◽  
Ionization Mass ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Lysine Residues

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7.

scholarly journals Ligand binding and heterodimerization with retinoid X receptor α (RXRα) induce farnesoid X receptor (FXR) conformational changes affecting coactivator binding

2018 ◽  
Vol 293 (47) ◽  
pp. 18180-18191 ◽  
Author(s):  
Na Wang ◽  
Qingan Zou ◽  
Jinxin Xu ◽  
Jiancun Zhang ◽  
Jinsong Liu
Keyword(s):  
Ligand Binding ◽  
Nuclear Receptor ◽  
Conformational Changes ◽  
Cholesterol Metabolism ◽  
Retinoid X Receptor ◽  
Farnesoid X Receptor ◽  
Dietary Fats ◽  
Structural Basis ◽  
Biliary Cirrhosis ◽  
C Terminus

Nuclear receptor farnesoid X receptor (FXR) functions as the major bile acid sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. Because of its central role in metabolism, FXR represents an important drug target to manage metabolic and other diseases, such as primary biliary cirrhosis and nonalcoholic steatohepatitis. FXR and nuclear receptor retinoid X receptor α (RXRα) form a heterodimer that controls the expression of numerous downstream genes. To date, the structural basis and functional consequences of the FXR/RXR heterodimer interaction have remained unclear. Herein, we present the crystal structures of the heterodimeric complex formed between the ligand-binding domains of human FXR and RXRα. We show that both FXR and RXR bind to the transcriptional coregulator steroid receptor coactivator 1 with higher affinity when they are part of the heterodimer complex than when they are in their respective monomeric states. Furthermore, structural comparisons of the FXR/RXRα heterodimers and the FXR monomers bound with different ligands indicated that both heterodimerization and ligand binding induce conformational changes in the C terminus of helix 11 in FXR that affect the stability of the coactivator binding surface and the coactivator binding in FXR. In summary, our findings shed light on the allosteric signal transduction in the FXR/RXR heterodimer, which may be utilized for future drug development targeting FXR.

scholarly journals Michler’s hydrol blue elucidates structural differences in prion strains

2020 ◽  
Vol 117 (47) ◽  
pp. 29677-29683
Author(s):  
Yiling Xiao ◽  
Sandra Rocha ◽  
Catherine C. Kitts ◽  
Anna Reymer ◽  
Tamás Beke-Somfai ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Density Functional ◽  
Amyloid Fibrils ◽  
Site Specific ◽  
Yeast Prions ◽  
Structural Details ◽  
Structural Differences ◽  
Environmentally Sensitive ◽  
A Site

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler’s hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

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