protein fold
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2022 ◽  
Author(s):  
Rachael Coleman ◽  
Melissa Bollmeyer ◽  
Sean Majer ◽  
Silas Ferrao ◽  
Kyle Lancaster

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide as part of the biogeochemical nitrogen cycle. They bear unique “heme P460” cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, This proenzyme undergoes complete maturation to active enzyme with spectroscopic properties that match wild-type cyt P460. Together, these data indicate that the cofactor is primed to undergo this covalent modification by virtue of the protein fold. A putative mechanism analogous to that used by heme oxygenases to degrade hemes is proposed.


Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2532
Author(s):  
Abid Javed ◽  
Hugo Villanueva ◽  
Shadikejiang Shataer ◽  
Sara Vasciaveo ◽  
Renos Savva ◽  
...  

Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and human infections that are especially lethal in newborns. Such pathogens are targeted by the obligate lytic phages of the Salasmaviridae and Guelinviridae families. These phages have relatively small ~20 kb linear protein-capped genomes and their compact organisation, relatively few structural elements, and broad host range, are appealing from a phage-engineering standpoint. In this study, we focus on portal proteins, which are core elements for the assembly of such tailed phages. The structures of dodecameric portal complexes from Salasmaviridae phage GA1, which targets Bacillus pumilus, and Guelinviridae phage phiCPV4 that infects Clostridium perfringens, were determined at resolutions of 3.3 Å and 2.9 Å, respectively. Both are found to closely resemble the related phi29 portal protein fold. However, the portal protein of phiCPV4 exhibits interesting differences in the clip domain. These structures provide new insights on structural diversity in Caudovirales portal proteins and will be essential for considerations in phage structural engineering.


2021 ◽  
Author(s):  
Molly J. McBride ◽  
Mrutyunjay A. Nair ◽  
Debangsu Sil ◽  
Jeffrey W. Slater ◽  
Monica Neugebauer ◽  
...  

ABSTRACTThe enzyme BesC from the β-ethynyl-L-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-L-lysine (produced from L-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-L-allylglycine and can analogously fragment L-Lys itself. BesC belongs to the emerging family of O2-activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here we show that binding of L-Lys or an analog triggers capture of O2 by the protein’s diiron(II) cofactor to form a blue µ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino-N-oxygenase domain of SznF. The ∼ 5- and ∼ 30-fold faster decay of the intermediate in reactions with 4-thia-L-Lys and (4RS)-chloro-DL-lysine than in the reaction with L-Lys itself, and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of L-allylglycine in the reaction with 4,4,5,5-[2H]-L-Lys, imply that the peroxide intermediate or a successor complex with which it reversibly interconverts initiates the oxidative fragmentation by abstracting hydrogen from C4. Surprisingly, the sluggish substrate L-Lys can dissociate after triggering the intermediate to form, thereby allowing one of the better substrates to bind and react. Observed linkage between Fe(II) and substrate binding suggests that the triggering event involves a previously documented (in SznF) ordering of the dynamic HDO architecture that contributes one of the iron sites, a hypothesis consistent with the observation that the diiron(III) product cluster produced upon decay of the intermediate spontaneously degrades, as it has been shown to do in all other HDOs studied to date.


2021 ◽  
Author(s):  
Monique K Merchant ◽  
Carlos Perez Mata ◽  
Yangci Liu ◽  
Haoming Zhai ◽  
Anna V Protasio ◽  
...  

Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus - an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum, with an envelope protein genetically related to GN-GC glycoproteins from phleboviruses. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. RNA-Seq data analysis detected transcripts mapping to Atlas virus at different stages of hookworm development. With its preserved biological activities, Atlas GC has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses.


2021 ◽  
Vol 17 (11) ◽  
pp. e1009620
Author(s):  
Xingjie Pan ◽  
Tanja Kortemme

A major challenge in designing proteins de novo to bind user-defined ligands with high affinity is finding backbones structures into which a new binding site geometry can be engineered with high precision. Recent advances in methods to generate protein fold families de novo have expanded the space of accessible protein structures, but it is not clear to what extend de novo proteins with diverse geometries also expand the space of designable ligand binding functions. We constructed a library of 25,806 high-quality ligand binding sites and developed a fast protocol to place (“match”) these binding sites into both naturally occurring and de novo protein families with two fold topologies: Rossman and NTF2. Each matching step involves engineering new binding site residues into each protein “scaffold”, which is distinct from the problem of comparing already existing binding pockets. 5,896 and 7,475 binding sites could be matched to the Rossmann and NTF2 fold families, respectively. De novo designed Rossman and NTF2 protein families can support 1,791 and 678 binding sites that cannot be matched to naturally existing structures with the same topologies, respectively. While the number of protein residues in ligand binding sites is the major determinant of matching success, ligand size and primary sequence separation of binding site residues also play important roles. The number of matched binding sites are power law functions of the number of members in a fold family. Our results suggest that de novo sampling of geometric variations on diverse fold topologies can significantly expand the space of designable ligand binding sites for a wealth of possible new protein functions.


2021 ◽  
Author(s):  
Neha Dhimole ◽  
Susanne Zur Lage ◽  
Wilfried Klug ◽  
Teresa Carlomagno

piRNAs are essential for transposon repression and protecting the germline from deleterious mutations. piRNA biogenesis comprises a primary and secondary pathway, and involves PIWI clade argonaute proteins and ancillary factors. Secondary piRNA biogenesis is tightly coupled to transposon repression. It requires processing of the 3-prime end of pre-piRNA during an amplification loop by an as yet unidentified endonuclease. Here, using crystallography, and biochemical assays, we discover that the Drosophila Qin protein, which is a critical member of the core amplification complex, has endonuclease activity. Qin contains five extended Tudor domains, which had been proposed to recognize methylated ligands. Instead, we show that these domains act as RNA-specific nucleases. This supports a role for Qin in the 3-prime end processing of Ago3-bound pre-piRNAs. Extended Tudor domains are frequent in piRNA-processing proteins, suggesting that the uncovered nuclease activity of this protein fold may be key to understanding the piRNA biogenesis.


2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Amelia Villegas-Morcillo ◽  
Victoria Sanchez ◽  
Angel M. Gomez

Abstract Background Current state-of-the-art deep learning approaches for protein fold recognition learn protein embeddings that improve prediction performance at the fold level. However, there still exists aperformance gap at the fold level and the (relatively easier) family level, suggesting that it might be possible to learn an embedding space that better represents the protein folds. Results In this paper, we propose the FoldHSphere method to learn a better fold embedding space through a two-stage training procedure. We first obtain prototype vectors for each fold class that are maximally separated in hyperspherical space. We then train a neural network by minimizing the angular large margin cosine loss to learn protein embeddings clustered around the corresponding hyperspherical fold prototypes. Our network architectures, ResCNN-GRU and ResCNN-BGRU, process the input protein sequences by applying several residual-convolutional blocks followed by a gated recurrent unit-based recurrent layer. Evaluation results on the LINDAHL dataset indicate that the use of our hyperspherical embeddings effectively bridges the performance gap at the family and fold levels. Furthermore, our FoldHSpherePro ensemble method yields an accuracy of 81.3% at the fold level, outperforming all the state-of-the-art methods. Conclusions Our methodology is efficient in learning discriminative and fold-representative embeddings for the protein domains. The proposed hyperspherical embeddings are effective at identifying the protein fold class by pairwise comparison, even when amino acid sequence similarities are low.


2021 ◽  
Vol 17 (9) ◽  
pp. e1008882
Author(s):  
Pablo Galaz-Davison ◽  
Ernesto A. Román ◽  
César A. Ramírez-Sarmiento

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weijing Gu ◽  
Shujuan Gao ◽  
Huan Wang ◽  
Kaelin D. Fleming ◽  
Reece M. Hoffmann ◽  
...  

AbstractPhospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


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