Sequence conservation and structural features that are common within TRP channels

TRP proteins are a large family of cation selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state of the channel are still missing for TRP channels. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed bioinformatic analyses over a large set of TRP channel genes. Here we report a discrete and exceptionally conserved set of residues. This fingerprint is composed of eleven residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbors the apparent differences in coupling strategies used by TRP subgroups.

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Self-association configures the NAD+-binding site of plant NLR TIR domains

10.1101/2021.10.02.462850 ◽

2021 ◽

Author(s):

Hayden Burdett ◽

Xiahao Hu ◽

Maxwell X Rank ◽

Natsumi Maruta ◽

Bostjan Kobe

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Active Site ◽

Binding Sites ◽

Molecular Mechanisms ◽

Structural Features ◽

Active Conformation ◽

Effector Triggered Immunity ◽

Self Association ◽

Tir Domains

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the ″AE interface″ (mediated by helices A and E), the ″BB-loop″ (connecting β-strand B and helix B in the structure), and the ″BE interface″ (mediated by the BB-loop from one TIR and the ″DE surface″ of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact ″DE surface″ is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

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Force transduction creates long-ranged coupling in ribosomes stalled by arrest peptides

10.1101/2020.10.16.342899 ◽

2020 ◽

Author(s):

Matthew H Zimmer ◽

Michiel JM Niesen ◽

Thomas F Miller

Keyword(s):

Amino Acids ◽

Secondary Structure ◽

Conformational Changes ◽

Protein Biosynthesis ◽

Essential Amino Acids ◽

Structural Features ◽

Applied Force ◽

Catalytic Center ◽

Force Propagation ◽

Force Transduction

AbstractForce-sensitive arrest peptides regulate protein biosynthesis by stalling the ribosome as they are translated. Synthesis can be resumed when the nascent arrest peptide experiences a pulling force of sufficient magnitude to break the stall. Efficient stalling is dependent on the specific identity of a large number of amino acids, including amino acids which are tens of angstroms away from the peptidyl transferase center (PTC). The mechanism of force-induced restart and the role of these essential amino acids far from the PTC is currently unknown. We use hundreds of independent molecular dynamics trajectories spanning over 120 μs in combination with kinetic analysis to characterize the barriers along the force-induced restarting pathway for the arrest peptide SecM. We find that the essential amino acids far from the PTC play a major role in controlling the transduction of applied force. In successive states along the stall-breaking pathway, the applied force propagates up the nascent chain until it reaches the C-terminus of SecM and the PTC, inducing conformational changes that allow for restart of translation. A similar mechanism of force propagation through multiple states is observed in the VemP stall-breaking pathway, but secondary structure in VemP allows for heterogeneity in the order of transitions through intermediate states. Results from both arrest peptides explain how residues that are tens of angstroms away from the catalytic center of the ribosome impact stalling efficiency by mediating the response to an applied force and shielding the amino acids responsible for maintaining the stalled state of the PTC.Significance StatementAs nascent proteins are synthesized by the ribosome, their interactions with the environment can create pulling forces on the nascent protein that can be transmitted to the ribosome’s catalytic center. These forces can affect the rate and even the outcome of translation. We use simulations to characterize the pathway of force transduction along arrest peptides and discover how secondary structure in the nascent protein and its interactions with the ribosome exit tunnel impede force propagation. This explains how amino acids in arrest peptides that are tens of angstroms away from the ribosome’s catalytic center contribute to stalling, and, more broadly, suggests how structural features in the nascent protein dictate the ribosome’s ability to functionally respond to its environment.

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Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation

10.1101/2021.04.21.440662 ◽

2021 ◽

Author(s):

Samuel J Craven ◽

Samson G.F. Condon ◽

Gladys Diaz-Vazquez ◽

Qiang Cui ◽

Alessandro Senes

Keyword(s):

Amino Acids ◽

Cell Wall ◽

Cell Division ◽

Conformational Changes ◽

Structural Model ◽

Coiled Coil ◽

Fine Tuning ◽

Marginal Stability ◽

Structural Element ◽

The Core

The FtsLB complex is a critical regulator of bacterial cell division, acting as a switch that modulates cell wall reconstruction. Evidence indicates that FtsLB exists in either an off or on state which supports the corresponding activation state of the peptidoglycan synthase complex FtsWI. In Escherichia coli, residues within FtsLB that are critical for this activation are located in a region near the C-terminal end of the periplasmic coiled coil, raising questions about the precise role of this conserved domain in the mechanism. Here, we investigate an unusual cluster of polar amino acids occurring within the core of the coiled coil. These amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is an intentionally "detuned" structural element. In addition, suppressor mutations were identified within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. Based on energetic and sequence propensity considerations, we propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the polar amino acids participate in packing within the core, but their hydrophilic terminal moieties remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). The Y-model remains well structured during molecular dynamics simulations, unlike the I-model, and satisfies all known experimental constraints. For this reason, we propose the Y-model as the configuration of the coiled coil of FtsLB and that a shift in this architecture, dependent on its marginal stability, is involved in activating the complex during the process that triggers septal cell wall reconstruction.

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A combined coarse-grained and all-atom simulation of TRPV1 channel gating and heat activation

The Journal of General Physiology ◽

10.1085/jgp.201411335 ◽

2015 ◽

Vol 145 (5) ◽

pp. 443-456 ◽

Cited By ~ 25

Author(s):

Wenjun Zheng ◽

Feng Qin

Keyword(s):

Conformational Changes ◽

Md Simulation ◽

Molecular Mechanisms ◽

Transient Receptor Potential ◽

Trp Channels ◽

Normal Mode Analysis ◽

Coarse Grained ◽

Mode Analysis ◽

Structural Dynamic ◽

Heat Activation

The transient receptor potential (TRP) channels act as key sensors of various chemical and physical stimuli in eukaryotic cells. Despite years of study, the molecular mechanisms of TRP channel activation remain unclear. To elucidate the structural, dynamic, and energetic basis of gating in TRPV1 (a founding member of the TRPV subfamily), we performed coarse-grained modeling and all-atom molecular dynamics (MD) simulation based on the recently solved high resolution structures of the open and closed form of TRPV1. Our coarse-grained normal mode analysis captures two key modes of collective motions involved in the TRPV1 gating transition, featuring a quaternary twist motion of the transmembrane domains (TMDs) relative to the intracellular domains (ICDs). Our transition pathway modeling predicts a sequence of structural movements that propagate from the ICDs to the TMDs via key interface domains (including the membrane proximal domain and the C-terminal domain), leading to sequential opening of the selectivity filter followed by the lower gate in the channel pore (confirmed by modeling conformational changes induced by the activation of ICDs). The above findings of coarse-grained modeling are robust to perturbation by lipids. Finally, our MD simulation of the ICD identifies key residues that contribute differently to the nonpolar energy of the open and closed state, and these residues are predicted to control the temperature sensitivity of TRPV1 gating. These computational predictions offer new insights to the mechanism for heat activation of TRPV1 gating, and will guide our future electrophysiology and mutagenesis studies.

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Conservation of Amino Acids into Multiple Alignments Involved in Pairwise Interactions in Three-Dimensional Protein Structures

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720003000228 ◽

2003 ◽

Vol 01 (03) ◽

pp. 505-520 ◽

Cited By ~ 1

Author(s):

Mounir Errami ◽

Christophe Geourjon ◽

Gilbert Deléage

Keyword(s):

Amino Acids ◽

Disulfide Bonds ◽

Hydrophobic Interactions ◽

Protein Structures ◽

Three Dimensional ◽

Structural Features ◽

Multiple Alignment ◽

Salt Bridges ◽

Multiple Alignments ◽

Multiple Sequences

We present an original strategy, that involves a bioinformatic software structure, in order to perform an exhaustive and objective statistical analysis of three-dimensional structures of proteins. We establish the relationship between multiple sequences alignments and various structural features of proteins. We show that amino acids implied in disulfide bonds, salt bridges and hydrophobic interactions are particularly conserved. Effects of identity, global similarity within alignments, and accessibility of interactions have been studied. Furthermore, we point out that the more variable the sequences within a multiple alignment, the more informative the multiple alignment. The results support multiple alignments usefulness for predictions of structural features.

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Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

10.1101/2020.08.31.275875 ◽

2020 ◽

Author(s):

Andrea Tagliani ◽

Jacopo Rossi ◽

Christophe H. Marchand ◽

Marcello De Mia ◽

Daniele Tedesco ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Protein S ◽

Structural Features ◽

Zinc Ion ◽

Kinetic Properties ◽

Coordination Environment ◽

Enzymatic Systems ◽

Catalytic Zinc

ABSTRACTProtein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. The kinetic properties of CrGSNOR1 were determined and the high-resolution three-dimensional structures of the apo and NAD+-bound forms of the enzyme were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved 3D-folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.One-sentence summaryGSNOR1 from Chlamydomonas reinhardtii displays an unusual variability of the catalytic zinc coordination environment and an unexpected resistance to thiol-based redox modifications

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Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

Science ◽

10.1126/science.1221863 ◽

2012 ◽

Vol 336 (6089) ◽

pp. 1708-1711 ◽

Cited By ~ 90

Author(s):

Corey S. Westfall ◽

Chloe Zubieta ◽

Jonathan Herrmann ◽

Ulrike Kapp ◽

Max H. Nanao ◽

...

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Molecular Basis ◽

Biochemical Analysis ◽

Plant Hormone ◽

Three Dimensional ◽

Structural Basis ◽

Hormone Action ◽

Plant Signaling ◽

Carboxyl Terminal

Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid–specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

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Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens

Biochemical Journal ◽

10.1042/bcj20160932 ◽

2017 ◽

Vol 474 (2) ◽

pp. 231-246 ◽

Cited By ~ 15

Author(s):

Leonor Morgado ◽

Marta Bruix ◽

P. Raj Pokkuluri ◽

Carlos A. Salgueiro ◽

David L. Turner

Keyword(s):

Conformational Changes ◽

Solution Structure ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Axial Ligand ◽

Geobacter Sulfurreducens ◽

Cellular Growth ◽

Dimensional Structure ◽

Structural Basis ◽

Natural Habitats

The periplasmic triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant; it is the likely reservoir of electrons to the outer surface to assist the reduction of extracellular terminal acceptors; these include insoluble metal oxides in natural habitats and electrode surfaces from which electricity can be harvested. A detailed thermodynamic characterization of PpcA showed that it has an important redox-Bohr effect that might implicate the protein in e−/H+ coupling mechanisms to sustain cellular growth. This functional mechanism requires control of both the redox state and the protonation state. In the present study, isotope-labeled PpcA was produced and the three-dimensional structure of PpcA in the oxidized form was determined by NMR. This is the first solution structure of a G. sulfurreducens cytochrome in the oxidized state. The comparison of oxidized and reduced structures revealed that the heme I axial ligand geometry changed and there were other significant changes in the segments near heme I. The pH-linked conformational rearrangements observed in the vicinity of the redox-Bohr center, both in the oxidized and reduced structures, constitute the structural basis for the differences observed in the pKa values of the redox-Bohr center, providing insights into the e−/H+ coupling molecular mechanisms driven by PpcA in G. sulfurreducens.

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Selectively Receptor-Blind Measles Viruses: Identification of Residues Necessary for SLAM- or CD46-Induced Fusion and Their Localization on a New Hemagglutinin Structural Model

Journal of Virology ◽

10.1128/jvi.78.1.302-313.2004 ◽

2004 ◽

Vol 78 (1) ◽

pp. 302-313 ◽

Cited By ~ 145

Author(s):

Sompong Vongpunsawad ◽

Numan Oezgun ◽

Werner Braun ◽

Roberto Cattaneo

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Measles Virus ◽

Structural Model ◽

Three Dimensional ◽

Lymphocyte Activation ◽

Cell Receptor ◽

Dimensional Model ◽

Three Dimensional Model ◽

H Protein

ABSTRACT Measles virus (MV) enters cells either through the signaling lymphocyte activation molecule SLAM (CD150) expressed only in immune cells or through the ubiquitously expressed regulator of complement activation, CD46. To identify residues on the attachment protein hemagglutinin (H) essential for fusion support through either receptor, we devised a strategy based on analysis of morbillivirus H-protein sequences, iterative cycles of mutant protein production followed by receptor-based functional assays, and a novel MV H three-dimensional model. This model uses the Newcastle disease virus hemagglutinin-neuraminidase protein structure as a template. We identified seven amino acids important for SLAM- and nine for CD46 (Vero cell receptor)-induced fusion. The MV H three-dimensional model suggests (i) that SLAM- and CD46-relevant residues are located in contiguous areas in propeller β-sheets 5 and 4, respectively; (ii) that two clusters of SLAM-relevant residues exist and that they are accessible for receptor contact; and (iii) that several CD46-relevant amino acids may be shielded from direct receptor contacts. It appears likely that certain residues support receptor-specific H-protein conformational changes. To verify the importance of the H residues identified with the cell-cell fusion assays for virus entry into cells, we transferred the relevant mutations into genomic MV cDNAs. Indeed, we were able to recover recombinant viruses, and we showed that these replicate selectively in cells expressing SLAM or CD46. Selectively receptor-blind viruses will be used to study MV pathogenesis and may have applications for the production of novel vaccines and therapeutics.

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Structure of DnmZ, a nitrososynthase in the Streptomyces peucetius anthracycline biosynthetic pathway

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15014272 ◽

2015 ◽

Vol 71 (10) ◽

pp. 1205-1214 ◽

Cited By ~ 2

Author(s):

Lauren Sartor ◽

Charmaine Ibarra ◽

Ahmad Al-Mestarihi ◽

Brian O. Bachmann ◽

Jessica L. Vey

Keyword(s):

Substrate Specificity ◽

Natural Product ◽

Crystal Structures ◽

Conformational Changes ◽

Biosynthetic Pathway ◽

Three Dimensional ◽

Structural Features ◽

Dimensional Structure ◽

X Ray ◽

X Ray Crystallography

The anthracyclines are a class of highly effective natural product chemotherapeutics and are used to treat a range of cancers, including leukemia. The toxicity of the anthracyclines has stimulated efforts to further diversify the scaffold of the natural product, which has led to renewed interest in the biosynthetic pathway responsible for the formation and modification of this family of molecules. DnmZ is an N-hydroxylating flavin monooxygenase (a nitrososynthase) that catalyzes the oxidation of the exocyclic amine of the sugar nucleotide dTDP-L-epi-vancosamine to its nitroso form. Its specific role in the anthracycline biosynthetic pathway involves the synthesis of the seven-carbon acetal moiety attached to C4 of L-daunosamine observed in the anthracycline baumycin. Here, X-ray crystallography was used to elucidate the three-dimensional structure of DnmZ. Two crystal structures of DnmZ were yielded: that of the enzyme alone, solved to 3.00 Å resolution, and that of the enzyme in complex with thymidine diphosphate, the nucleotide carrier portion of the substrate, solved to 2.74 Å resolution. These models add insights into the structural features involved in substrate specificity and conformational changes involved in thymidine diphosphate binding by the nitrososynthases.

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