scholarly journals Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

2021 ◽  
Author(s):  
Julien Lescar ◽  
Side Hu ◽  
Abbas El Sahili ◽  
Srujana Kishore ◽  
Xinya Hemu ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Reaction ◽  
Mechanistic Explanation ◽  
Structural Basis ◽  
Hydrophobic Residue ◽  
Vacuolar Compartment ◽  
Molecular Events ◽  
Activated State ◽  
Limited Class

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the substrate P2' position. Beside illustrating the role played by P1 and P2' residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities.

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

scholarly journals Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains

2017 ◽  
Author(s):  
Marie Morrow ◽  
Michael Morgan ◽  
Marcello Clerici ◽  
Katerina Growkova ◽  
Ming Yan ◽  
...  
Keyword(s):  
Active Site ◽  
Tight Binding ◽  
Dominant Negative ◽  
Structural Basis ◽  
Deubiquitinating Enzymes ◽  
Active Site Cysteine ◽  
Binding Domains ◽  
Common Strategy ◽  
Catalytic Cysteine

ABSTRACTA common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

scholarly journals Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA

2021 ◽  
Author(s):  
Gabriela Garcia-Rodriguez ◽  
Daniel Charlier ◽  
Dorien Wilmaerts ◽  
Jan Michiels ◽  
Remy Loris
Keyword(s):  
Active Site ◽  
Resting State ◽  
Protein Function ◽  
Structural Basis ◽  
Conformational Heterogeneity ◽  
Defense System ◽  
Nucleotide Binding Domain ◽  
Dimer Interface ◽  
Higher Eukaryotes

ABSTRACTThe rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was recently identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

Convergence of the SUMO and MAPK pathways on the ETS-domain transcription factor Elk-1

2006 ◽  
Vol 73 ◽  
pp. 121-129 ◽  
Author(s):  
Shen-Hsi Yang ◽  
Andrew D. Sharrocks
Keyword(s):  
Transcription Factor ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Highly Active ◽  
Molecular Events ◽  
Extracellular Signal ◽  
Activated State ◽  
Mitogen Activated Protein ◽  
Ets Domain

The ETS-domain transcription factor Elk-1 is regulated by phosphorylation in response to activation of the MAPK (mitogen-activated protein kinase) pathways. This phosphorylation triggers a series of molecular events that convert Elk-1 from a transcriptionally silent state into a highly active state and then back to a basal level. At the same time, activation of the ERK (extracellular-signal-regulated kinase) MAPK pathway leads to loss of modification of Elk-1 by SUMO (small ubiquitin-related modifier). As SUMO imparts repressive properties on Elk-1, ERK-mediated SUMO loss leads to de-repression at the same time as the ERK pathway promotes activation of Elk-1. Thus a two-step mechanism is employed to convert Elk-1 into its fully activated state. Here, the molecular events underlying these changes in Elk-1 status, and the role of PIASxα [protein inhibitor of activated STAT (signal transducer and activator of transcription) xα] as a co-activator that facilitates this process, are discussed.

scholarly journals Structural basis of the complementary activity of two ketosynthases in aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Jiwon Yeon ◽  
Eunha Hwang ◽  
...  
Keyword(s):  
Active Site ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
Aryl Group ◽  
Vitro Assay ◽  
Active Site Cavity

AbstractAryl polyenes (APE) are one of the most widespread secondary metabolites among gram-negative bacteria. In Acinetobacter baumannii, strains belonging to the virulent global clone 2 (GC2) mostly contain APE biosynthesis genes; its relevance in elevated pathogenicity is of great interest. APE biosynthesis gene clusters harbor two ketosynthases (KSs): the heterodimeric KS-chain length factor complex, ApeO-ApeC, and the homodimeric ketoacyl-acyl carrier protein synthase I (FabB)-like KS, ApeR. The role of the two KSs in APE biosynthesis is unclear. We determined the crystal structures of the two KSs from a pathogenic A. baumannii strain. ApeO-ApeC and ApeR have similar cavity volumes; however, ApeR has a narrow cavity near the entrance. In vitro assay based on the absorption characteristics of polyene species indicated the generation of fully elongated polyene with only ApeO-ApeC, probably because of the funnel shaped active site cavity. However, adding ApeR to the reaction increases the throughput of APE biosynthesis. Mutagenesis at Tyr135 in the active site cavity of ApeR reduces the activity significantly, which suggests that the stacking of the aryl group between Tyr135 and Phe202 is important for substrate recognition. Therefore, the two KSs function complementarily in the generation of APE to enhance its production.

scholarly journals Structural basis and mechanism of the unfolding-induced activation of an acid response chaperone HdeA

2018 ◽  
Author(s):  
Xing-Chi Yu ◽  
Yunfei Hu ◽  
Jienv Ding ◽  
Hongwei Li ◽  
Changwen Jin
Keyword(s):  
Chemical Exchange ◽  
Structural Disorder ◽  
Experimental Information ◽  
Activation Mechanism ◽  
Chemical Exchange Saturation Transfer ◽  
Structural Basis ◽  
Activation Process ◽  
Chaperone Function ◽  
Activated State

ABSTRACTThe role of protein structural disorder in biological functions is gaining increasing interests in the past decade. The bacterial acid-resistant chaperone HdeA belongs to a group of “conditionally disordered” protein that is activated via an order-to-disorder transition. However, the mechanism for unfolding-induced activation remains unclear due to the lack of experimental information on the unfolded state conformation and the chaperone-client interactions. Here we use advanced solution NMR methods to characterize the activated state conformation of HdeA under acidic condition and identify the client binding sites. The activated HdeA becomes largely disordered and exposes two essential hydrophobic patches of residues for client interactions. The pH-dependent chemical exchange saturation transfer (CEST) result identifies three acid-sensitive regions that act as structural locks during the activation process, revealing a multi-step activation mechanism of HdeA chaperone function at atomic level. The results highlight the role of protein disorder in chaperone function and the self-inhibitory role of ordered structures under non-stress conditions, offering new insights for further understanding the protein structure-function paradigm.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX)4 and Coumarin-RXDX Conjugate β-Sheets

2020 ◽  
Author(s):  
Ryan Weber ◽  
Martin McCullagh
Keyword(s):  
Biological Imaging ◽  
Ph Sensing ◽  
Hydrophobic Residue ◽  
Ideal Candidate ◽  
Self Assembling ◽  
Sensing Applications ◽  
Β Sheets ◽  
The Stability ◽  
Dynamics Simulations

<p>pH-switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X=Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX)<sub>4</sub> systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X=Ala are found to be unstable and both pHs while conjugates with X=Val, Leu, Ile and Phe are found to form stable β-sheets at least at neutral pH. The (RFDF)<sub>4</sub>-coumarin conjugate is found to have the largest relative entropy value of 0.884 +/- 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF)<sub>4</sub> containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.</p>

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