Biology subjects

Alvarez-Carreno, C.

Publications and source records attributed to Alvarez-Carreno, C..

7 recordsLinked to original sources

bioRxiv2026

TEDLH: Domain HMMs for sensitive detection of remote homologues

MotivationThe Encyclopedia of Domains (TED) provides domain annotations for proteins in the AlphaFold Protein Structure Database (AFDB) using a consensus of three state-of-the-art structure-based methods. We used these TED domain annotations to construct profile Hidden Markov models (HMMs), collectively forming the TED Library of HMMs (TEDLH). TEDLH enables sensitive sequence and profile searches, supporting systematic exploration of protein domain families and their evolutionary relationships. ResultsTEDLH links domain HMMs to experimentally determined CATH-PDB structures through direct (primary) and transitive (secondary and tertiary) relationships. Fewer than half of TEDLH HMMs are directly linked to a CATH-PDB domain; the remaining models are connected through transitive relationships. These transitive links extend coverage into more divergent regions of sequence space and better represent CATH superfamily diversity. HMM-HMM comparisons within CATH superfamily 3.30.70.100 illustrate how transitive relationships expand sequence coverage in TEDLH. In this superfamily, 4,813 TEDLH HMMs are connected to 212 CATH-PDB representatives. Primary, secondary, and tertiary relationships progressively capture more divergent sequences (pairwise sequence identity <20%) that retain structural similarity (TM-score >0.6) and a conserved two-layer /{beta} sandwich core fold. All-against-all HMM-HMM comparisons across TEDLH also reveal sequence similarities across the CATH hierarchy (cross-hits). At low query coverage (<50%), cross-hits are more frequent between CATH classes, whereas at higher coverage thresholds (>70%) they predominantly occur between superfamilies. These cross-hits are not driven by superfamily size or sequence diversity and can provide guidance for CATH curation. As an example, analysis of cross-hits between superfamilies 2.170.130.30 and 3.10.20.30 reveals evolutionary relationships between these groups. Availability and ImplementationTEDLH is compatible with HH-suite3 and is available from FigShare https://doi.org/10.6084/m9.figshare.28531754 for local use. Contactc.carreno@ucl.ac.uk

bioinformatics↗

bioRxiv2024

AARS Online: a collaborative database on the structure, function, and evolution of the aminoacyl-tRNA synthetases

The aminoacyl-tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various non-translational activities, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein. Across the tree of life, the twenty-two coded amino acids are handled by sixteen evolutionary Families of Class I aaRS and twenty-one Families of Class II aaRS. We introduce AARS Online, an interactive Wikipedia-like tool curated by an international consortium of field experts. This platform systematizes existing knowledge about the aaRS by showcasing a taxonomically diverse selection of aaRS sequences and structures. Through its graphical user interface, AARS Online facilitates a seamless exploration between protein sequence and structure, providing a friendly introduction to the material for non-experts and a useful resource for experts. Curated multiple sequence alignments can be extracted for downstream analyses. Accessible at www.aars.online, AARS Online is a free resource to delve into the world of the aaRS.

biochemistry↗

bioRxiv2024

BEAN and HABAS: Polyphyletic insertions in RNAP that point to deep evolutionary divergence of bacteria

The {beta} and {beta} subunits of the RNA polymerase (RNAP) are large proteins with complex multi-domain architectures that include several insertional domains. Here, we analyze the multi-domain organizations of bacterial RNAP-{beta} and RNAP-{beta} using sequence, experimentally determined structures and AlphaFold structure predictions. We observe that bacterial lineage-specific domains in RNAP-{beta} belong to a group of domains that we call BEAN (Broadly Embedded ANnex) and that in RNAP-{beta}, bacterial lineage-specific domains are HAmmerhead/BArrel-Sandwich Hybrid (HABAS) domains. The BEAN domain has a characteristic three-dimensional structure composed of two square bracket-like elements that are antiparallel relative to each other. The HABAS domain contains a four-stranded open {beta}-sheet with a GD-box-like motif in one of the {beta}-strands and the adjoining loop. The BEAN domain is identified not only in the bacterial RNAP-{beta}, but also in the archaeal version of universal ribosomal protein L10. The HABAS domain is observed as an insertional domain in several metabolic proteins. The phylogenetic distributions of bacterial lineage-specific insertional domains of {beta} and {beta} subunits of RNAP follow the Tree of Life. The presence of insertional domains can help establish a relative timeline of events in the evolution of a protein because insertion is inferred to post-date the base domain. We discuss mechanisms that might account for the discovery of homologous insertional domains in non-equivalent locations in bacteria and archaea.

evolutionary biology↗

bioRxiv2023

Common evolutionary origins of the bacterial glycyl tRNA synthetaseand alanyl tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA-modifying proteins. Component domains of bacGlyRS show common ancestry with i) the catalytic domain of class II tRNA synthetases; ii) the HD domain of the bacterial RNase Y; iii) the body and tail domains of the archaeal CCA-adding enzyme; iv) the anti-codon binding domain of the arginyl tRNA synthetase; and v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain is found only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined data suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.

evolutionary biology↗

bioRxiv2023

Translation in a Box: Orthogonal Evolution in the Saccharomyces cerevisiae Mitochondrion

The ability to re-engineer and creatively evolve the translation system (TS) would allow invention of new coded polymers by altering the amino acid sidechain inventory and by shifting the polypeptide backbone into new chemical spaces. Unfortunately, the TS is difficult to manipulate and is more constrained over evolution than any other biological system. An orthogonal TS, running in parallel to the primary TS within a given host cell, would release constraints and allow TS manipulation. A fully orthogonal TS requires dedicated rRNAs, rProteins, aminoacyl-tRNA synthetases, and initiation and termination factors, none of which interact with the primary TS. The S. cerevisiae mitochondrial TS is fully orthogonal to the cytosolic TS. Mito-rRNAs, mito- rProteins, mito-tRNAs, mito-aminoacyl tRNA synthetases, and mito-translation factors are distinct from, physically separated from, and functionally independent of their cytosolic counterparts. Here, the S. cerevisiae mitochondrial translation system was subjected to various stresses including antibiotics, mutagenesis and truncation of mito-rProteins, or wholesale replacement of mito-rProteins. Directed evolution of these stressed systems was facilitated by controlled transitions between fermentation and respiration, by changing the carbon source in the growth medium; the dependence of S. cerevisiae survival on mitochondrial translation can be toggled on and off. Specific recreation of the resulting mutations recapitulate the evolved phenotypes. The method developed here appears to be a general approach for discovering functional dependencies. Suppressor mutations reveal functional dependencies within the S. cerevisiae mitochondrial TS. For example proteins Rrg9 or Mrx1 interact with the mito-TS and have critical role in its function. The combined results indicate that the S. cerevisiae mitochondrial TS can be engineered and evolved in isolation of the cytosolic TS. SignificanceThe Central Dogma of Molecular Biology rules life on Earth. Information flows from DNA to mRNA to protein. In the last step of the Central Dogma, the translation system decodes mRNA and produces coded proteins by linking amino acids into polymers. Engineering and evolving the translation system could permits full technical control over this process and could lead to the generation of novel polymers. Here, we use the mitochondrial translation system in the budding yeast Saccharomyces cerevisiae for directed evolution of translation. We modify and evolve the translation system both directly and indirectly using antibiotics and gene editing tools and then measure resulting functionality. Our results show this secondary translation system inside S. cerevisiae mitochondria can be used as an approach for translation engineering.

synthetic biology↗

bioRxiv2022

Fold Evolution by Creative Destruction

Mechanisms by which new protein folds emerge and diverge pose central questions in biological sciences. Incremental mutation and step-wise adaptation explain relationships between topologically similar protein folds. However, the universe of folds is diverse and riotous, suggesting roles of more potent and creative forces. Sequence and structure similarity are observed between topologically distinct folds, indicating that proteins with distinct folds may share common ancestry. We found evidence of common ancestry between three distinct {beta}-barrel folds: OB, SH3 and cradle loop barrel (CLB). The data suggest a mechanism of fold evolution that interconverts SH3, OB and CLB. This mechanism, which we call creative destruction, can be generalized to explain other examples of fold evolution including circular permutation. In creative destruction, an open reading frame duplicates or otherwise merges with another to produce a fused polypeptide. A merger forces two ancestral domains into a new sequence and spatial context. The fused polypeptide can explore folding landscapes that are inaccessible to either of the independent ancestral domains. However, the folding landscapes of the fused polypeptide are not fully independent of those of the ancestral domains. Creative destruction is thus partially conservative in that a daughter fold would inherit some motifs from the ancestral folds. After a merger and refolding, adaptive processes such as mutation and loss of extraneous segments optimize the new daughter fold. SignificanceMechanisms of emergence and early diversification of structured proteins present deep and difficult problems in evolutionary biology. Here we excavate the deepest evolutionary history, found within the translation machinery, which is an ancient molecular fossil and the birthplace of all proteins. We demonstrate common origins of some of the simplest, oldest and most common protein folds. Furthermore, the data suggest a mechanism, that we call creative destruction, that explains at molecular level how simple folds spawn more complex folds. In this mechanism, new folds emerge from old folds via gene duplication, expression, exploration of new folding landscapes and adaptation. Creative destruction explains the facile emergence of complex from simple architectures in a funneled exploration.

evolutionary biology↗

bioRxiv2021

An updated structural model of the A domain of the Pseudomonas putida XylR regulator exposes a distinct interplay with aromatic effectors

A revised model of the aromatic binding A domain of the {sigma}54-dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR, and DmpR. The resulting frame was instrumental for mapping the large number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic-binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc-binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Proteomic analyses of the thereby tagged peptides confirmed the predicted in/out distribution of residues but also suggested that the fully-folded protein is not accessible by externally added effectors. The data thus suggested that XylR inducers assist the folding and/or the structuring of the A domain in an intramolecular non-repressive form rather than interacting dynamically with the aromatic partner once a fully structured protein is shaped. Originality-Significance StatementXylR is a transcriptional regulator of Pseudomonas putida strain mt-2 which activates the upper TOL pathway promoter Pu for catabolism of toluene and m-xylene upon binding of these aromatic effectors to its N-terminal A domain. While this feature has made XylR a popular platform for the development of whole-cell biosensors for aromatic compounds, the difficulty to crystallize the A domain --let alone the whole-length protein-- has made structural comprehension of the effector-regulator binding quite problematic. To overcome this impasse, we have combined homology-based structural predictions of the A domain of XylR with biochemical probing of exposed amino acids on the surface of the protein, both in vivo and in vitro. The results generally matched the effects of mutations known from previous genetic/phenotypic analyses of the protein. However, the data also suggested an intriguing mechanism of activation of XylR by effectors in which the inducer assists the shaping of the regulator in an active conformation rather than interacting a posteriori with an already formed protein invitro. This may in fact explain the longstanding failure to purify the protein in an effector-responsive form.

microbiology↗