scholarly journals Dormant spores sense amino acids through the B subunits of their germination receptors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lior Artzi ◽  
Assaf Alon ◽  
Kelly P. Brock ◽  
Anna G. Green ◽  
Amy Tam ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Binding Pocket ◽  
Nutrient Sensing ◽  
Membrane Receptors ◽  
Variation Analysis ◽  
Family Function ◽  
Ligand Binding Pocket ◽  
B Subunits

AbstractBacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection.

scholarly journals Broad Specificity of Amino Acid Chemoreceptor CtaA of Pseudomonas fluorescens Is Afforded by Plasticity of Its Amphipathic Ligand-Binding Pocket

2020 ◽  
Vol 33 (4) ◽  
pp. 612-623 ◽  
Author(s):  
Abu I. M. S. Ud-Din ◽  
Mohammad F. Khan ◽  
Anna Roujeinikova
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ligand Binding ◽  
Pseudomonas Fluorescens ◽  
Binding Pocket ◽  
Signaling Mechanism ◽  
Ligand Binding Pocket ◽  
Plant Growth Promoting ◽  
Chemotaxis Receptors ◽  
Broad Specificity

Motile bacteria follow gradients of nutrients or other environmental cues. Many bacterial chemoreceptors that sense exogenous amino acids contain a double Cache (dCache; calcium channels and chemotaxis receptors) ligand-binding domain (LBD). A growing number of studies suggest that broad-specificity dCache-type receptors that sense more than one amino acid are common. Here, we present an investigation into the mechanism by which the dCache LBD of the chemoreceptor CtaA from a plant growth–promoting rhizobacterium, Pseudomonas fluorescens, recognizes several chemically distinct amino acids. We established that amino acids that signal by directly binding to the CtaA LBD include ones with aliphatic (l-alanine, l-proline, l-leucine, l-isoleucine, l-valine), small polar (l-serine), and large charged (l-arginine) side chains. We determined the structure of CtaA LBD in complex with different amino acids, revealing that its ability to recognize a range of structurally and chemically distinct amino acids is afforded by its easily accessible plastic pocket, which can expand or contract according to the size of the ligand side chain. The amphipathic character of the pocket enables promiscuous interactions with both polar and nonpolar amino acids. The results not only clarify the means by which various amino acids are recognized by CtaA but also reveal that a conserved mobile lid over the ligand-binding pocket adopts the same conformation in all complexes, consistent with this being an important and invariant part of the signaling mechanism.

scholarly journals Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

2015 ◽  
Vol 90 (2) ◽  
pp. 1062-1069 ◽  
Author(s):  
Dana N. Raugi ◽  
Robert A. Smith ◽  
Geoffrey S. Gottlieb ◽  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ligand Binding ◽  
Protease Inhibitors ◽  
Binding Pocket ◽  
Wild Type ◽  
Ligand Binding Pocket ◽  
Crystallographic Structures ◽  
Retroviral Replication ◽  
Hiv 1

ABSTRACTProtease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2.IMPORTANCEProteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine FDA-approved HIV-1 protease inhibitors (PI) are active against HIV-2. The underlying reasons for intrinsic PI resistance in HIV-2 are not known. We examined the contributions of four amino acids in the ligand-binding pocket of the enzyme that differ between HIV-1 and HIV-2 by constructing HIV-2 clones encoding the corresponding HIV-1 amino acids and testing the PI susceptibilities of the resulting viruses. We found that the HIV-2 clone containing all four changes (PRΔ4) was as susceptible as HIV-1 to all nine PI. We also modeled the PRΔ4 enzyme structure and compared it to existing crystallographic structures of HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir. Our findings demonstrate that four positions in the ligand-binding cleft of protease are the primary cause of HIV-2 PI resistance.

scholarly journals SUN-LB138 Dynamic Structural Model of Testosterone Entry Into the Unliganded Androgen Receptor

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Irina Krylova ◽  
Fred J Schaufele ◽  
Christophe Guilbert
Keyword(s):  
Amino Acids ◽  
Androgen Receptor ◽  
Ligand Binding ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Binding Pocket ◽  
Binding Domain ◽  
Receptor Ligand ◽  
Ligand Binding Pocket ◽  
Crystallographic Structures

Abstract Background: Crystallographic structures of nuclear receptor ligand binding domains provide a static model of a receptor stably wrapped around an internalized ligand. Understanding the dynamics of a receptor at different stages of ligand binding has been hampered by the paucity of crystal structures for unliganded nuclear receptors. Molecular dynamic models have been constructed for some nuclear receptors to fill that void. Methods: The molecular simulation docking program MORDOR (MOlecular Recognition with a Driven dynamics OptimizeR)(1) was used to study the structural dynamics of the androgen receptor ligand binding domain (AR LBD) modeled from the static structure of the AR LBD bound to testosterone (T) (PDB ID: 2AM9). The goals of the study were to understand a) the dynamic interaction of the T in its binding pocket, b) AR LBD structural flexibilities that permit T entry/exit from the binding pocket and c) a model of the unliganded AR LBD. Results: Modeling AR LBD structure flexibility over time revealed possible alternative dynamic structures, including those without ligand, overlaid against the canonical nuclear receptor structure. The model dynamically tracks the structural changes as a ligand enters into the ligand binding domain and nestles into the ligand binding pocket. The model predicted the appearance of alpha helices within the AR LBD that transiently fold/unfold during the ligand entry phases. Once in the pocket, the ligand itself remains very dynamic in a still flexible pocket. The model predicted also AR LBD amino acids that sequentially interact with the ligand during its dynamic entry into the AR LBD. Intriguingly, those AR amino acids include those mutated in castration-resistant prostate tumors that continue to grow during androgen suppression therapy. Functional studies showed those mutant ARs had a primary consequence of enhancing response to lower level T, and other androgens, consistent with their role in creating a higher affinity AR that can scavenge low-level androgens in an androgen-suppressed patient. Conclusions: The molecular model of T binding to the AR LBD suggests a degree of structural dynamism not evident in the crystallographic structures commonly associated with nuclear receptors. Some AR mutations activating prostate tumor growth may do so by impacting androgen entry/exit, rather than by altering androgen fit into the ligand binding pocket. Reference: (1) Guilbert C, James TL (2008) J Chem Inf Model. 2008 48(6): 1257-1268. doi: 10.1021/ci8000327

scholarly journals Minireview: Nutrient Sensing by G Protein-Coupled Receptors

2013 ◽  
Vol 27 (8) ◽  
pp. 1188-1197 ◽  
Author(s):  
Eric M. Wauson ◽  
Andrés Lorente-Rodríguez ◽  
Melanie H. Cobb
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Taste Receptor ◽  
Sweet Taste ◽  
Nutrient Sensing ◽  
G Protein Coupled Receptors ◽  
Amino Acid Availability ◽  
Class C ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.

scholarly journals Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

2010 ◽  
Vol 76 (5) ◽  
pp. 1653-1660 ◽  
Author(s):  
Ponnandy Prabhu ◽  
Marimuthu Jeya ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Catalytic Efficiency ◽  
Homology Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
High Turnover ◽  
Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

Structural Coupling of 11-cis-7-Methyl-retinal and Amino Acids at the Ligand Binding Pocket of Rhodopsin

2009 ◽  
Vol 85 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Mònica Aguilà ◽  
Darwin Toledo ◽  
Margarita Morillo ◽  
Marta Dominguez ◽  
Belén Vaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
Binding Pocket ◽  
Structural Coupling ◽  
Ligand Binding Pocket

scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

scholarly journals Sequence of the N-terminal half of Bacillus amyloliquefaciens α-amylase

1980 ◽  
Vol 185 (2) ◽  
pp. 387-395 ◽  
Author(s):  
H Chung ◽  
F Friedberg
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Primary Structure ◽  
Bacillus Amyloliquefaciens ◽  
Proteolytic Enzymes ◽  
Alpha Amylase ◽  
Sequence Determination ◽  
N Terminus

Bacillus amyloliquefaciens alpha-amylase (1,4-alpha-D-glucan glucanohydrolase. EC 3.2.1.1), which is commercially supplied as ‘Bacillus subtilis alpha-amylase’ does not cross-react immunologically with B. subtilis alpha-amylase. This enzyme (from B. amyloliquefaciens) was cleaved by treatment with CNBr into seven fragments. Peptide A was selected for sequence determination. It is the longest one, containing 185 amino acids (i.e. approx. 50% of the total molecule) and connects to the hexapeptide of the N-terminus. Its primary structure was aligned by use of various proteolytic enzymes. The sequence of amino acids 181-184 is identical with that of amino acids 14-17 of the alpha-amylase isolated from B. subtilis (except that amino acid 183 is asparagine rather than aspartic acid).

scholarly journals Postprandial nutrient-sensing and metabolic responses after partial dietary fishmeal replacement by soyabean meal in turbot (Scophthalmus maximus L.)

2015 ◽  
Vol 115 (3) ◽  
pp. 379-388 ◽  
Author(s):  
Dandan Xu ◽  
Gen He ◽  
Kangsen Mai ◽  
Huihui Zhou ◽  
Wei Xu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nutrient Retention ◽  
Scophthalmus Maximus ◽  
Amino Acid Profile ◽  
Nutrient Sensing ◽  
Fish Growth ◽  
Metabolic Responses ◽  
Amino Acid Supplementation ◽  
Soyabean Meal

AbstractIn this study, we chose a carnivorous fish, turbot (Scophthalmus maximus L.), to examine its nutrient-sensing and metabolic responses after ingestion of diets with fishmeal (FM), or 45 % of FM replaced by soyabean meal (34·6 % dry diet) balanced with or without essential amino acids (EAA) to match the amino acid profile of FM diet for 30 d. After a 1-month feeding trial, fish growth, feed efficiency and nutrient retention were markedly reduced by soyabean meal-incorporated (SMI) diets. Compared with the FM diet, SMI led to a reduction of postprandial influx of free amino acids, hypoactivated target of rapamycin signalling and a hyperactivated amino acid response pathway after refeeding, a status associated with reduced protein synthesis, impaired postprandial glycolysis and lipogenesis. These differential effects were not ameliorated by matching an EAA profile of soyabean meal to that of the FM diet through dietary amino acid supplementation. Therefore, this study demonstrated that the FM diet and SMI diets led to distinct nutrient-sensing responses, which in turn modulated metabolism and determined the utilisation efficiency of diets. Our results provide a new molecular explanation for the role of nutrient sensing in the inferior performance of aquafeeds in which FM is replaced by soyabean meal.

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