High Resolution Structural Insights into Heliorhodopsin Family

AbstractRhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes.

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High-resolution structural insights into the heliorhodopsin family

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915888117 ◽

2020 ◽

Vol 117 (8) ◽

pp. 4131-4141 ◽

Cited By ~ 8

Author(s):

K. Kovalev ◽

D. Volkov ◽

R. Astashkin ◽

A. Alekseev ◽

I. Gushchin ◽

...

Keyword(s):

Schiff Base ◽

Bioinformatic Analysis ◽

Proton Acceptor ◽

Water Molecules ◽

Primary Proton ◽

Biological Processes ◽

New Family ◽

Charged Amino Acids ◽

Base Cavity ◽

Structural Insights

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

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Integrated bioinformatic analysis of HNF1A in human cancers

Journal of International Medical Research ◽

10.1177/0300060521997326 ◽

2021 ◽

Vol 49 (3) ◽

pp. 030006052199732

Author(s):

Enfan Zhang ◽

Xi Huang ◽

Jingsong He

Keyword(s):

Immune Cells ◽

Prognostic Value ◽

Bioinformatic Analysis ◽

Nuclear Factor ◽

Biological Processes ◽

Hepatocyte Nuclear Factor ◽

Patients With Cancer ◽

Kaplan Meier ◽

And Function ◽

Kaplan Meier Plotter

Objectives Cancer is a threat to human health, and many molecules are involved in the transformation of malignant cells. Hepatocyte nuclear factor 1A (HNF1A) is an important transcription factor that regulates multiple biological processes. Our research focused on elucidating the expression and function of HNF1A in cancer through bioinformatic analysis. Methods UALCAN, Kaplan–Meier plotter, COSMIC, Tumor IMmune Estimation Resource, and Cancer Regulome were used to obtain relevant data for HNF1A. Results HNF1A was abnormally expressed in multiple cancers, and its expression was associated with differences in overall survival in patients with cancer. HNF1A mutations widely exist in tumors and interact with different genes involved in various processes. Additionally, we found that HNF1A was associated with the infiltration of immune cells, and it affected the prognostic value of these cells in some cancers. Conclusions HNF1A plays a crucial role in cancer, and it may represent a biomarker and target for future cancer immunotherapy.

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Structural insights into the molecular mechanism of the m6A writer complex

eLife ◽

10.7554/elife.18434 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 151

Author(s):

Paweł Śledź ◽

Martin Jinek

Keyword(s):

Molecular Mechanism ◽

Active Site ◽

Evolutionary History ◽

Rna Binding ◽

Biological Processes ◽

Catalytic Core ◽

Genome Regulation ◽

History Of ◽

Structural Insights ◽

Critical Aspects

Methylation of adenosines at the N6 position (m6A) is a dynamic and abundant epitranscriptomic mark that regulates critical aspects of eukaryotic RNA metabolism in numerous biological processes. The RNA methyltransferases METTL3 and METTL14 are components of a multisubunit m6A writer complex whose enzymatic activity is substantially higher than the activities of METTL3 or METTL14 alone. The molecular mechanism underpinning this synergistic effect is poorly understood. Here we report the crystal structure of the catalytic core of the human m6A writer complex comprising METTL3 and METTL14. The structure reveals the heterodimeric architecture of the complex and donor substrate binding by METTL3. Structure-guided mutagenesis indicates that METTL3 is the catalytic subunit of the complex, whereas METTL14 has a degenerate active site and plays non-catalytic roles in maintaining complex integrity and substrate RNA binding. These studies illuminate the molecular mechanism and evolutionary history of eukaryotic m6A modification in post-transcriptional genome regulation.

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Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms22052647 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2647

Author(s):

M. Quadir Siddiqui ◽

Maulik D. Badmalia ◽

Trushar R. Patel

Keyword(s):

Nucleic Acid ◽

Nuclear Export ◽

Consensus Sequence ◽

Nuclear Export Signal ◽

Bioinformatic Analysis ◽

Nucleic Acid Binding ◽

Protein Protein Interaction ◽

Lim Domains ◽

Protein Nucleic Acid ◽

And Function

Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and functions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share similarities with transcriptional regulators and have positively charged electrostatic patches, which may indicate that they have previously unanticipated nucleic acid binding properties. Intrinsic dynamics analysis of Lim domains suggest that only Lim1 has similar internal dynamics properties, unlike Lim2/3. Furthermore, we analyzed protein expression and mutational frequency in various malignancies, as well as mapped protein-protein interaction networks they are involved in. Overall, our comprehensive bioinformatic analysis suggests that these proteins may play important roles in mediating protein-protein and protein-nucleic acid interactions.

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HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

Nature Communications ◽

10.1038/s41467-021-21302-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Fa-Hui Sun ◽

Peng Zhao ◽

Nan Zhang ◽

Lu-Lu Kong ◽

Catherine C. L. Wong ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Active Site ◽

Negative Charge ◽

Structural Data ◽

Dna Breaks ◽

Adp Ribosylation ◽

Local Conformation ◽

Key Residues ◽

Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

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Fe(2)OG: an integrated HMM profile-based web server to predict and analyze putative non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenase function in protein sequences

BMC Research Notes ◽

10.1186/s13104-021-05477-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Siddhartha Kundu

Keyword(s):

Amino Acid ◽

Water Molecule ◽

Active Site ◽

Ferrous Iron ◽

Web Server ◽

Protein Sequences ◽

Diverse Group ◽

And Function ◽

Functionally Diverse ◽

Haem Iron

Abstract Objective Non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenases (i2OGdd), are a taxonomically and functionally diverse group of enzymes. The active site comprises ferrous iron in a hexa-coordinated distorted octahedron with the apoenzyme, 2-oxoglutarate and a displaceable water molecule. Current information on novel i2OGdd members is sparse and relies on computationally-derived annotation schema. The dissimilar amino acid composition and variable active site geometry thereof, results in differing reaction chemistries amongst i2OGdd members. An additional need of researchers is a curated list of sequences with putative i2OGdd function which can be probed further for empirical data. Results This work reports the implementation of $$Fe\left(2\right)OG$$ F e 2 O G , a web server with dual functionality and an extension of previous work on i2OGdd enzymes $$\left(Fe\left(2\right)OG\equiv \{H2OGpred,DB2OG\}\right)$$ F e 2 O G ≡ { H 2 O G p r e d , D B 2 O G } . $$Fe\left(2\right)OG$$ F e 2 O G , in this form is completely revised, updated (URL, scripts, repository) and will strengthen the knowledge base of investigators on i2OGdd biochemistry and function. $$Fe\left(2\right)OG$$ F e 2 O G , utilizes the superior predictive propensity of HMM-profiles of laboratory validated i2OGdd members to predict probable active site geometries in user-defined protein sequences. $$Fe\left(2\right)OG$$ F e 2 O G , also provides researchers with a pre-compiled list of analyzed and searchable i2OGdd-like sequences, many of which may be clinically relevant. $$Fe(2)OG$$ F e ( 2 ) O G , is freely available (http://204.152.217.16/Fe2OG.html) and supersedes all previous versions, i.e., H2OGpred, DB2OG.

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Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin

Nature Communications ◽

10.1038/s41467-021-24429-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Stephan Hirschi ◽

David Kalbermatter ◽

Zöhre Ucurum ◽

Thomas Lemmin ◽

Dimitrios Fotiadis

Keyword(s):

Proton Translocation ◽

Functional Characterization ◽

Green Light ◽

Proton Donor ◽

Molecular Organization ◽

Primary Proton ◽

Proton Pumps ◽

Model Protein ◽

Dynamics Simulations ◽

And Function

AbstractThe green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

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Dynamics and mechanism of ultrafast water–protein interactions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602916113 ◽

2016 ◽

Vol 113 (30) ◽

pp. 8424-8429 ◽

Cited By ~ 77

Author(s):

Yangzhong Qin ◽

Lijuan Wang ◽

Dongping Zhong

Keyword(s):

Protein Interactions ◽

Hydration Shell ◽

Water Dynamics ◽

Side Chain ◽

Water Molecules ◽

Ultrafast Dynamics ◽

Hydration Water ◽

Water Side ◽

Dynamics And Function ◽

And Function

Protein hydration is essential to its structure, dynamics, and function, but water–protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship.

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Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

Journal of Virology ◽

10.1128/jvi.01158-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 6

Author(s):

Wenhua Kuang ◽

Huanyu Zhang ◽

Manli Wang ◽

Ning-Yi Zhou ◽

Fei Deng ◽

...

Keyword(s):

Enzymatic Activity ◽

Active Site ◽

Salt Bridge ◽

Sulfhydryl Oxidase ◽

Dimer Interface ◽

Conserved Regions ◽

Occlusion Bodies ◽

Budded Virus ◽

And Function ◽

Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

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