scholarly journals Integrative modelling of the full-length human dehydrodolichyl diphosphate synthase using a hybrid computational and experimental approach

2019 ◽  
Author(s):  
Michal Lisnyansky Barel ◽  
Su Youn Lee ◽  
Ah Young Ki ◽  
Noa Kapelushnik ◽  
Anat Loewenstein ◽  
...  
Keyword(s):  
Structural Model ◽  
Clinical Importance ◽  
Protein Glycosylation ◽  
Full Length ◽  
Scattering Data ◽  
Exchange Mass Spectrometry ◽  
Functional Studies ◽  
Terminal Domain ◽  
Helix Loop Helix ◽  
Structural Framework

AbstractDehydrodolichyl diphosphate synthase (DHDDS) and Nogo-B receptor (NgBR) form the heteromeric human cis-prenyltransferase complex, synthesizing the precursor for the glycosyl carrier involved in N-linked protein glycosylation. In line with the important role of N-glycosylation in protein biogenesis, mutations in DHDDS, the catalytic subunit of the complex, were shown to result in human diseases. Importantly, well-characterized DHDDS homologs function as homodimers and not as heteromeric complexes. Moreover, DHDDS encompasses a C-terminal region, which does not converge with any known conserved domains. Therefore, despite the clinical importance of DHDDS, our understating of its structure-function relations remains poor. Here, we provide a structural model for the full-length human DHDDS using a multidisciplinary experimental and computational approach. Our model suggests that the C-terminal domain of DHDDS forms a helix-loop-helix motif, tightly packed against the core catalytic cis-prenyltransferase domain. This model is consistent with small-angle X-ray scattering data, indicating that the full-length DHDDS maintains a similar conformation in solution. Moreover, hydrogen-deuterium exchange mass-spectrometry experiments show time-dependent deuterium uptake in the C-terminal domain, consistent with its overall folded state. Finally, we provide a model for the DHDDS-NgBR heterodimer, offering a structural framework for future structural and functional studies of the human cis-prenyltransferase complex.

scholarly journals Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 660 ◽  
Author(s):  
Michal Lisnyansky Bar-El ◽  
Su Youn Lee ◽  
Ah Young Ki ◽  
Noa Kapelushnik ◽  
Anat Loewenstein ◽  
...  
Keyword(s):  
Structural Model ◽  
Catalytic Domain ◽  
Clinical Importance ◽  
Protein Glycosylation ◽  
Full Length ◽  
Size Exclusion ◽  
Scattering Data ◽  
Exchange Mass Spectrometry ◽  
Functional Studies ◽  
C Terminus

Dehydrodolichyl diphosphate synthase (DHDDS) is the catalytic subunit of the heteromeric human cis-prenyltransferase complex, synthesizing the glycosyl carrier precursor for N-linked protein glycosylation. Consistent with the important role of N-glycosylation in protein biogenesis, DHDDS mutations result in human diseases. Importantly, DHDDS encompasses a C-terminal region, which does not converge with any known conserved domains. Therefore, despite the clinical importance of DHDDS, our understating of its structure–function relations remains poor. Here, we provide a structural model for the full-length human DHDDS using a multidisciplinary experimental and computational approach. Size-exclusion chromatography multi-angle light scattering revealed that DHDDS forms a monodisperse homodimer in solution. Enzyme kinetics assays revealed that it exhibits catalytic activity, although reduced compared to that reported for the intact heteromeric complex. Our model suggests that the DHDDS C-terminus forms a helix–turn–helix motif, tightly packed against the core catalytic domain. This model is consistent with small-angle X-ray scattering data, indicating that the full-length DHDDS maintains a similar conformation in solution. Moreover, hydrogen–deuterium exchange mass-spectrometry experiments show time-dependent deuterium uptake in the C-terminal domain, consistent with its overall folded state. Finally, we provide a model for the DHDDS–NgBR heterodimer, offering a structural framework for future structural and functional studies of the complex.

scholarly journals Structural analyses of the Haemophilus influenzae peptidoglycan synthase activator LpoA suggest multiple conformations in solution

2017 ◽  
Vol 292 (43) ◽  
pp. 17626-17642 ◽  
Author(s):  
Karthik Sathiyamoorthy ◽  
J. Vijayalakshmi ◽  
Bhramara Tirupati ◽  
Lixin Fan ◽  
Mark A. Saper
Keyword(s):  
Haemophilus Influenzae ◽  
Outer Membrane ◽  
Normal Mode ◽  
Normal Mode Analysis ◽  
Full Length ◽  
Scattering Data ◽  
Mode Analysis ◽  
Bacterial Viability ◽  
Terminal Domain ◽  
Multiple Conformations

In many Gram-negative bacteria, the peptidoglycan synthase PBP1A requires the outer membrane lipoprotein LpoA for constructing a functional peptidoglycan required for bacterial viability. Previously, we have shown that the C-terminal domain of Haemophilus influenzae LpoA (HiLpoA) has a highly conserved, putative substrate-binding cleft between two α/β lobes. Here, we report a 2.0 Å resolution crystal structure of the HiLpoA N-terminal domain. Two subdomains contain tetratricopeptide-like motifs that form a concave groove, but their relative orientation differs by ∼45° from that observed in an NMR structure of the Escherichia coli LpoA N domain. We also determined three 2.0–2.8 Å resolution crystal structures containing four independent full-length HiLpoA molecules. In contrast to an elongated model previously suggested for E. coli LpoA, each HiLpoA formed a U-shaped structure with a different C-domain orientation. This resulted from both N-domain twisting and rotation of the C domain (up to 30°) at the end of the relatively immobile interdomain linker. Moreover, a previously predicted hinge between the lobes of the LpoA C domain exhibited variations of up to 12°. Small-angle X-ray scattering data revealed excellent agreement with a model calculated by normal mode analysis from one of the full-length HiLpoA molecules but even better agreement with an ensemble of this molecule and two of the partially extended normal mode analysis-predicted models. The different LpoA structures helped explain how an outer membrane-anchored LpoA can either withdraw from or extend toward the inner membrane-bound PBP1A through peptidoglycan gaps and hence regulate the synthesis of peptidoglycan necessary for bacterial viability.

scholarly journals An integrative structural model of the full-length gp16 ATPase in bacteriophage phi29 DNA packaging motor

2020 ◽  
Author(s):  
Abdullah F.U.H. Saeed ◽  
Chun Chan ◽  
Hongxin Guan ◽  
Bing Gong ◽  
Peixuan Guo ◽  
...  
Keyword(s):  
Structural Model ◽  
Md Simulations ◽  
Full Length ◽  
Nucleic Acid Binding ◽  
Multiple Sources ◽  
Cellular Functions ◽  
Motor Complex ◽  
Terminal Domain ◽  
Binding Surface ◽  
The Individual

ABSTRACTBiological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. Most of these biomotors belong to a group of enzymes known as ATPases, which adopt a multi-subunit ring-shaped structure and hydrolyze adenosine triphosphate (ATP) to generate forces. The gene product 16 (gp16), an ATPase in bacteriophage □29, is among the most powerful biomotors known. It can overcome substantial resisting forces from entropic, electrostatic, and DNA bending sources to package double-stranded DNA (dsDNA) into a preformed protein shell (procapsid). Despite numerous studies of the □29 packaging mechanism, a structure of the full-length gp16 is still lacking, let alone that of the packaging motor complex that includes two additional molecular components: a connector gp10 protein and a prohead RNA (pRNA). Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment of gp16-CTD with related RNase H-like nuclease domains revealed a nucleic acid binding surface in gp16-CTD, whereas no nuclease activity has been detected for gp16. Subsequent molecular dynamics (MD) simulations showed that this nucleic acid binding surface is likely essential for pRNA binding. Furthermore, our simulations of a full-length gp16 structural model highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid, providing structural support to the previously proposed inchworm model. Lastly, we assembled an atomic structural model of the complete □29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage □29 and suggested how the individual domains of gp16 work together to power such translocation.ABSTRACT (SHORT)Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. The gene product 16 (gp16) in bacteriophage □29 is among the most powerful biomotors known, which adopts a multi-subunit ring-shaped structure and hydrolyzes ATP to package double-stranded DNA (dsDNA) into a preformed procapsid. Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment and molecular dynamics (MD) simulations revealed an essential binding surface of gp16-CTD for prohead RNA (pRNA), a unique component of the motor complex. Furthermore, our simulations highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid. Lastly, we assembled an atomic structural model of the complete □29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage □29 and suggested how the individual domains of gp16 work together to power such translocation.

scholarly journals Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

2016 ◽  
Vol 113 (30) ◽  
pp. E4357-E4366 ◽  
Author(s):  
Giambattista Guaitoli ◽  
Francesco Raimondi ◽  
Bernd K. Gilsbach ◽  
Yacob Gómez-Llorente ◽  
Egon Deyaert ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Structural Model ◽  
3D Structure ◽  
Kinase Domain ◽  
Full Length ◽  
Regulatory Domains ◽  
Catalytic Domains ◽  
Structural Framework ◽  
Kinase Domain Mutations

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.

scholarly journals Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 597
Author(s):  
Haoran Zhang ◽  
Qiuxiang Zhou ◽  
Chenyun Guo ◽  
Liubin Feng ◽  
Huilin Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ribosomal Protein ◽  
Solution Structure ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Hydrophobic Core ◽  
Terminal Domain ◽  
Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

scholarly journals Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3488
Author(s):  
Fuqiang Ban ◽  
Eric Leblanc ◽  
Ayse Derya Cavga ◽  
Chia-Chi Flora Huang ◽  
Mark R. Flory ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Splice Variants ◽  
Full Length ◽  
Cancer Resistance ◽  
Castration Resistant Prostate Cancer ◽  
Terminal Domain ◽  
Castration Resistant ◽  
Coregulatory Proteins ◽  
Androgen Binding

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

scholarly journals ADAMTS13 and 15 are not regulated by the full length and N-terminal domain forms of TIMP-1, -2, -3 and -4

2015 ◽  
Vol 4 (1) ◽  
pp. 73-78 ◽  
Author(s):  
CENQI GUO ◽  
ANASTASIA TSIGKOU ◽  
MENG HUEE LEE
Keyword(s):  
Full Length ◽  
Terminal Domain

scholarly journals A structural model of the Fábrica Nova region, Santa Rita syncline, Quadrilátero Ferrífero: flanking folds as a folding mechanism

2015 ◽  
Vol 68 (2) ◽  
pp. 153-162 ◽  
Author(s):  
Daniel Quinaud Rossi ◽  
Issamu Endo
Keyword(s):  
Structural Model ◽  
Axial Direction ◽  
Ore Deposits ◽  
Tectonic Deformation ◽  
Folding Mechanism ◽  
Structural Framework ◽  
Deformation Phase ◽  
Proposed Model ◽  
Quadrilátero Ferrífero ◽  
Iron Ore Deposits

AbstractThis study focuses on the eastern flank of the Santa Rita syncline (Dorr 1969), with specific emphasis on the region known as Fábrica Nova. Important iron ore deposits are located on the flanks of this structure, such as Timbopeba, Alegria, São Luiz, Tamanduá, Almas and Fábrica Nova. The Santa Rita syncline is a fold with N-S axial direction and of subregional scale, with roots in the adjacent basement of the Santa Bárbara Complex and sectioned by the Água Quente thrust fault. The hypothesis of this study is that the structural framework of the region resulted from the superposition of at least three deformation phases on the Ouro Preto nappe. The Fábrica Nova mine, located in the central portion of the study area, is embedded in a synformal structure with a 100/20 trending axis named Fábrica Nova synform. The proposed model to explain the particular structural geometry of this region is based on the flanking folding mechanism (Passchier 2001). This mechanism may have been developed by E-W crustal shortening during the F4 tectonic deformation phase.

scholarly journals Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

2020 ◽  
Author(s):  
Aniefon Ibuot ◽  
Rachel E. Webster ◽  
Lorraine E. Williams ◽  
Jon K. Pittman
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Metal Binding ◽  
Metal Tolerance ◽  
Alginate Beads ◽  
Full Length ◽  
Wild Type ◽  
Microalgal Biomass ◽  
Terminal Domain ◽  
Metal Biosorption ◽  
Transgenic Cells

AbstractThe use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalisation of both metals compared to wild type. AtHMA4 was expressed either as the full-length protein or just the C-terminal tail, which is known to have metal binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the full-length protein or C-terminal domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilisation in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilisation maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild type cells. This demonstrates that expressing AtHMA4 full-length or C-terminus has great potential as a strategy for bioremediation using microalgal biomass.

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