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Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. A major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid is often used as part of commercial rooting mixtures, yet many recalcitrant plants do not form ARs in response to this treatment. Here we describe the synthesis and screening of a focused library of synthetic auxin conjugates in Eucalyptus grandis cuttings and identify 4-chlorophenoxyacetic acid-L-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants, including apple and argan. Comprehensive metabolic and functional analyses reveal that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds for more effective plant growth regulation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Protein space is characterized by extensive recurrence, or "reuse," of parts, suggesting that new proteins and domains can evolve by mixing-and-matching of existing segments. From an evolutionary perspective, for a given combination to persist, the protein segments should presumably not only match geometrically but also dynamically communicate with each other to allow concerted motions that are key to function. Evidence from protein space supports the premise that domains indeed combine in this manner; we explore whether a similar phenomenon can be observed at the sub-domain level. To this end, we use Gaussian Network Models (GNMs) to calculate the so-called soft modes, or low-frequency modes of motion for a dataset of 150 protein domains. Modes of motion can be used to decompose a domain into segments of consecutive amino acids that we call "dynamic elements", each of which belongs to one of two parts that move in opposite senses. We find that, in many cases, the dynamic elements, detected based on GNM analysis, correspond to established "themes": Sub-domain-level segments that have been shown to recur in protein space, and which were detected in previous research using sequence similarity alone (i.e. completely independently of the GNM analysis). This statistically significant correlation hints at the importance of dynamics in evolution. Overall, the results are consistent with an evolutionary scenario where proteins have emerged from themes that need to match each other both geometrically and dynamically, e.g. to facilitate allosteric regulation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-β and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated β-strand sheets forming a cross-β configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-β amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR's conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric 'hotspot' located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric 'hotspot' that could serve as a promising target for the development of novel therapeutic interventions., Competing Interests: AE, BA, NL, NB, TH, OL No competing interests declared, (© 2023, Ersoy, Altintel, Livnat Levanon et al.)

Multiple sequence alignments (MSAs) are the workhorse of molecular evolution and structural biology research. From MSAs, the amino acids that are tolerated at each site during protein evolution can be inferred. However, little is known regarding the repertoire of tolerated amino acids in proteins when only a few or no sequence homologs are available, such as orphan and de novo designed proteins. Here we present EvoRator2, a deep-learning algorithm trained on over 15,000 protein structures that can predict which amino acids are tolerated at any given site, based exclusively on protein structural information mined from atomic coordinate files. We show that EvoRator2 obtained satisfying results for the prediction of position-weighted scoring matrices (PSSM). We further show that EvoRator2 obtained near state-of-the-art performance on proteins with high quality structures in predicting the effect of mutations in deep mutation scanning (DMS) experiments and that for certain DMS targets, EvoRator2 outperformed state-of-the-art methods. We also show that by combining EvoRator2's predictions with those obtained by a state-of-the-art deep-learning method that accounts for the information in the MSA, the prediction of the effect of mutation in DMS experiments was improved in terms of both accuracy and stability. EvoRator2 is designed to predict which amino-acid substitutions are tolerated in such proteins without many homologous sequences, including orphan or de novo designed proteins. We implemented our approach in the EvoRator web server (https://evorator.tau.ac.il)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Cation/proton antiporters (CPAs) regulate cells' salt concentration and pH. Their malfunction is associated with a range of human pathologies, yet only a handful of CPA-targeting therapeutics are presently in clinical development. Here, we discuss how recently published mammalian protein structures and emerging computational technologies may help to bridge this gap., Competing Interests: Declaration of interests None declared by the authors., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The ConSurf web-sever for the analysis of proteins, RNA, and DNA provides a quick and accurate estimate of the per-site evolutionary rate among homologues. The analysis reveals functionally important regions, such as catalytic and ligand-binding sites, which often evolve slowly. Since the last report in 2016, ConSurf has been improved in multiple ways. It now has a user-friendly interface that makes it easier to perform the analysis and to visualize the results. Evolutionary rates are calculated based on a set of homologous sequences, collected using hidden Markov model-based search tools, recently embedded in the pipeline. Using these, and following the removal of redundancy, ConSurf assembles a representative set of effective homologues for protein and nucleic acid queries to enable informative analysis of the evolutionary patterns. The analysis is particularly insightful when the evolutionary rates are mapped on the macromolecule structure. In this respect, the availability of AlphaFold model structures of essentially all UniProt proteins makes ConSurf particularly relevant to the research community. The UniProt ID of a query protein with an available AlphaFold model can now be used to start a calculation. Another important improvement is the Python re-implementation of the entire computational pipeline, making it easier to maintain. This Python pipeline is now available for download as a standalone version. We demonstrate some of ConSurf's key capabilities by the analysis of caveolin-1, the main protein of membrane invaginations called caveolae., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The opportunistic fungus Aspergillus fumigatus is the primary invasive mold pathogen in humans, and is responsible for an estimated 200,000 yearly deaths worldwide. Most fatalities occur in immunocompromised patients who lack the cellular and humoral defenses necessary to halt the pathogen's advance, primarily in the lungs. One of the cellular responses used by macrophages to counteract fungal infection is the accumulation of high phagolysosomal Cu levels to destroy ingested pathogens. A. fumigatus responds by activating high expression levels of crpA , which encodes a Cu + P-type ATPase that actively transports excess Cu from the cytoplasm to the extracellular environment. In this study, we used a bioinformatics approach to identify two fungal-unique regions in CrpA that we studied by deletion/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse model of invasive pulmonary aspergillosis. Deletion of CrpA fungal-unique amino acids 1-211 containing two N-terminal Cu-binding sites, moderately increased Cu-sensitivity but did not affect expression or localization to the endoplasmic reticulum (ER) and cell surface. Replacement of CrpA fungal-unique amino acids 542-556 consisting of an intracellular loop between the second and third transmembrane helices resulted in ER retention of the protein and strongly increased Cu-sensitivity. Deleting CrpA N-terminal amino acids 1-211 or replacing amino acids 542-556 also increased sensitivity to killing by mouse alveolar macrophages. Surprisingly, the two mutations did not affect virulence in a mouse model of infection, suggesting that even weak Cu-efflux activity by mutated CrpA preserves fungal virulence.

The human Na + /H + antiporter NHA2 (SLC9B2) transports Na + or Li + across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity., (© 2022 The Protein Society.)

Influenza A virus is the most virulent influenza subtype and is associated with large-scale global pandemics characterized by high levels of morbidity and mortality. Developing simple and sensitive molecular methods for detecting influenza viruses is critical. Neuraminidase, an exo-glycosidase displayed on the surface of influenza virions, is responsible for the release of the virions and their spread in the infected host. Here, we present a new phenoxy-dioxetane chemiluminescent probe (CLNA) that can directly detect neuraminidase activity. The probe exhibits an effective turn-on response upon reaction with neuraminidase and produces a strong emission signal at 515 nm with an extremely high signal-to-noise ratio. Comparison measurements of our new probe with previously reported analogous neuraminidase optical probes showed superior detection capability in terms of response time and sensitivity. Thus, as far as we know, our probe is the most sensitive neuraminidase probe known to date. The chemiluminescence turn-on response produced by our neuraminidase probe enables rapid screening for small molecules that inhibit viral replication through different mechanisms as validated directly in influenza A-infected mammalian cells using the known inhibitors oseltamivir and amantadine. We expect that our new chemiluminescent neuraminidase probe will prove useful for various applications requiring neuraminidase detection including drug discovery assays against various influenza virus strains in mammalian cells., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The emergence of novel proteins, beyond these that can be readily made by duplication and recombination of preexisting domains, is elusive. De novo emergence from random sequences is unlikely because the vast majority of random chains would not even fold, let alone function. An alternative explanation is that novel proteins emerge by duplication and fusion of pre-existing polypeptide segments. In this case, traces of such ancient events may remain within contemporary proteins in the form of reused segments. Together with the late Dan Tawfik, we detected such similar segments, far shorter than intact protein domains, which are found in different environments. The detection of these, "bridging themes," was based on a unique search strategy, where in addition to searching for similarity of shared fragments, so-called "themes," we also explicitly searched for cases in which the sequence segments before and after the theme are dissimilar (both in sequence and structure). Here, using a similar strategy, we further expanded the search and discovered almost 500 additional "bridging themes," linking domains that are often from ancient folds. The themes, of 20 residues or more (average 53), do not retain their structure despite sharing 37% sequence identity on average. Indeed, conformation flexibility may confer an evolutionary advantage, in that it fits in multiple environments. We elaborate on two interesting themes, shared between Rossmann/Trefoil-Plexin-like domains and a β-propeller-like domain. FOR A BROAD AUDIENCE: A fundamental question in molecular evolution is how protein domains emerged. Similar segments shared between domains of seemingly distinct origins, may offer clues, as these may be remnants of the evolutionary process through which these domains emerged. However, finding such cases is difficult. Here, we expand the set of such cases which we curated previously, adding segments shared between domains that are considered ancient., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Accurate protein structure predictors use clusters of homologues, which disregard sequence specific effects. In this issue of Structure, Weißenow and colleagues report a deep learning-based tool, EMBER2, that efficiently predicts the distances in a protein structure from its amino acid sequence only. This approach should enable the analysis of mutation effects., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Measuring evolutionary rates at the residue level is indispensable for gaining structural and functional insights into proteins. State-of-the-art tools for estimating rates take as input a large set of homologous proteins, a probabilistic model of evolution and a phylogenetic tree. However, a gap exists when only few or no homologous proteins can be found, e.g., orphan proteins. In addition, such tools do not take the three-dimensional (3D) structure of the protein into account. The association between the 3D structure and site-specific rates can be learned using machine-learning regression tools from a cohort of proteins for which both the structure and a large set of homologs exist. Here we present EvoRator, a user-friendly web server that implements a machine-learning regression algorithm to predict site-specific evolutionary rates from protein structures. We show that EvoRator outperforms predictions obtained using traditional physicochemical features, such as relative solvent accessibility and weighted contact number. We also demonstrate the application of EvoRator in three common scenarios that arise in protein evolution research: (1) orphan proteins for which no (or few) homologs exist; (2) When homologous sequences exist, our algorithm contrasts structure-based estimates of the evolutionary rates and the phylogeny-based estimates. This allows detecting sites that are likely conserved due to functional rather than structural constraints; (3) Algorithms that only rely on homologous sequence often fail to accurately measure the evolutionary rates of positions in gapped sequence alignments, which frequently occurs as a result of a clade-specific insertion. Our algorithm makes use of training data and known 3D structure of such gapped positions to predict their evolutionary rates. EvoRator is freely available for all users at: https://evorator.tau.ac.il/., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu 2+ ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu + and Cu 2+ with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)