scholarly journals Structural basis of heterotetrameric assembly and disease mutations in the human cis-prenyltransferase complex

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Michal Lisnyansky Bar-El ◽  
Pavla Vaňková ◽  
Adva Yeheskel ◽  
Luba Simhaev ◽  
Hamutal Engel ◽  
...  
Keyword(s):  
Active Site ◽  
Site Formation ◽  
Structural Basis ◽  
Functional Coupling ◽  
Synthesis Mechanism ◽  
Pt Complex ◽  
C Terminus ◽  
Disease Mutations ◽  
Functional Consequences ◽  
Dynamics Simulations

Abstract The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. Here, we reveal that hcis-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for hcis-PT dysfunction in retinitis pigmentosa. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

scholarly journals Structure of the human heterotetrameric cis-prenyltransferase complex

2020 ◽  
Author(s):  
Michal Lisnyansky Bar-El ◽  
Pavla Vankova ◽  
Petr Man ◽  
Yoni Haitin ◽  
Moshe Giladi
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Substrate Binding ◽  
Synthesis Mechanism ◽  
Allosteric Communication ◽  
Pt Complex ◽  
C Terminus ◽  
Length Determination ◽  
Enzymatic Complex

AbstractThe human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, we reveal here that hcis-PT exhibits a novel heterotetrameric assembly in solution, composed of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and two inactive Nogo-B receptor (NgBR) subunits. The 2.3 Å crystal structure of the complex exposes a dimer-of-heterodimers arrangement, with DHDDS C-termini serving as homotypic assembly domains. Furthermore, the structure elucidates the molecular details associated with substrate binding, catalysis, and product length determination. Importantly, the distal C-terminus of NgBR transverses across the heterodimeric interface, directly participating in substrate binding and underlying the allosteric communication between the subunits. Finally, mapping disease-associated hcis-PT mutations involved in blindness, neurological and glycosylation disorders onto the structure reveals their clustering around the active site. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

scholarly journals Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2018 ◽  
Vol 74 (12) ◽  
pp. 765-769 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Chi-Hung Huang ◽  
Shu-Jung Lai ◽  
Yeh Chen
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Peptidoglycan Synthesis ◽  
C Terminus ◽  
Dimeric Protein ◽  
Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

scholarly journals Structural basis for UFM1 transfer from UBA5 to UFC1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar ◽  
Prasanth Padala ◽  
Jamal Fahoum ◽  
Fouad Hassouna ◽  
Tomer Tsaban ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Basis ◽  
Adenylation Domain ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Processes ◽  
Enzyme Cascade ◽  
Linear Sequence ◽  
C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

scholarly journals Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

2022 ◽  
Author(s):  
Andrea Piserchio ◽  
Eta A Isiroho ◽  
Kimberly Long ◽  
Amanda L Bohanon ◽  
Eric A Kumar ◽  
...  
Keyword(s):  
Active Site ◽  
Protein Quality ◽  
Regulatory Element ◽  
Structural Basis ◽  
Calmodulin Binding ◽  
Intimate Association ◽  
Biochemical Studies ◽  
Functional Consequences ◽  
Heterodimeric Complex ◽  
Elongation Phase

Translation is a highly energy consumptive process tightly regulated for optimal protein quality and adaptation to energy and nutrient availability. A key facilitator of this process is the α-kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process mechanistically distinct from other calmodulin-dependent kinases. Here we resolve the atomic details of this mechanism through a 2.3 Å crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a spine that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

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.

scholarly journals Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Kristin Kassler ◽  
Julia Meier ◽  
Jutta Eichler ◽  
Heinrich Sticht
Keyword(s):  
Salt Bridge ◽  
Disulfide Bridge ◽  
Structural Basis ◽  
Binding Assays ◽  
Viral Glycoprotein ◽  
Gp120 Binding ◽  
C Terminus ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Hiv 1

The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4). Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120.

scholarly journals Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 907
Author(s):  
Bodee Nutho ◽  
Salila Pengthaisong ◽  
Anupong Tankrathok ◽  
Vannajan Sanghiran Lee ◽  
James R. Ketudat Cairns ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Binding Energy ◽  
Active Site ◽  
The Other ◽  
Strong Preference ◽  
Structural Basis ◽  
Active Site Residue ◽  
Weakly Bound ◽  
Sugar Moiety ◽  
Dynamics Simulations

β-Glucosidases and β-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 β-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the −1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant β-glycosidases.

scholarly journals Structural Basis for Autoinhibition of CTP:Phosphocholine Cytidylyltransferase (CCT), the Regulatory Enzyme in Phosphatidylcholine Synthesis, by Its Membrane-binding Amphipathic Helix

2013 ◽  
Vol 289 (3) ◽  
pp. 1742-1755 ◽  
Author(s):  
Jaeyong Lee ◽  
Svetla G. Taneva ◽  
Bryan W. Holland ◽  
D. Peter Tieleman ◽  
Rosemary B. Cornell
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Membrane Lipid ◽  
Structural Basis ◽  
Soluble Form ◽  
Amphipathic Helix ◽  
Inhibitory Interaction ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Membrane Bound ◽  
Dynamics Simulations

CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves disruption of an inhibitory interaction between the αE helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-αE interaction as a cluster of four amphipathic helices (two αE and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-αE interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device.

Structural basis and specificity of human otubain 1-mediated deubiquitination

2009 ◽  
Vol 418 (2) ◽  
pp. 379-390 ◽  
Author(s):  
Mariola J. Edelmann ◽  
Alexander Iphöfer ◽  
Masato Akutsu ◽  
Mikael Altun ◽  
Katalin di Gleria ◽  
...  
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Structural Basis ◽  
Cellular Interaction ◽  
Deubiquitinating Enzyme ◽  
Molecular Function ◽  
Guanine Nucleotide Binding Protein ◽  
Polyubiquitin Chains ◽  
C Terminus

OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys48-linked polyubiquitin chains over Lys63-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1′ site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys48-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein β polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.

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