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Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.

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The red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) is one of the most harmful pests for palm trees, causing serious economic damage worldwide. The present work aims to model and study the 3D structures of highly expressed odorant binding proteins from R. ferrugineus (RferOBPs) and identify possible binding modes and ligand release mechanism by docking and molecular dynamics. Highly confident 3D structures of a total of 11 odorant binding proteins (OBPs) were obtained with AlphaFold2. All 3D RferOBPs modeled structures displayed six characteristic α-helices, except for RfeOBP7 and RfeOBP10, which had an extra terminal α-helix. Among the eleven modeled RferOBPs, RferOBP4 was highly expressed in the antennae and subsequently selected for further analyses. Molecular docking analyses demonstrated that ferruginol, α-pinene, DEET, and picaridin can favorably bind the RferOBP4 cavity with low affinity energies. Molecular dynamic simulations of RferOBP4 bound to ferruginol at different pH values showed that low pH environments dictate a structural change into an apo-state that modifies the number of tunnels where the ligand can coexist, further triggering ligand release by a pH-dependent mechanism. This is the first report concerning the modelling and study of ligand binding modes and release mechanism of R. ferrugineus OBPs.Communicated by Ramaswamy H. Sarma.

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Beyond the problem in public health that protist-generated diseases represent, understanding the variety of mechanisms used by these parasites to interact with the human immune system is of biological and medical relevance. Giardia lamblia is an early divergent eukaryotic microorganism showing remarkable pathogenic strategies for evading the immune system of vertebrates. Among various multifunctional proteins in Giardia, arginine deiminase is considered an enzyme that plays multiple regulatory roles during the life cycle of this parasite. One of its most important roles is the crosstalk between the parasite and host. Such a molecular "chat" is mediated in human cells by membrane receptors called Toll-like receptors (TLRs). Here, we studied the importance of the 3D structure of giardial arginine deiminase (GlADI) to immunomodulate the human immune response through TLRs. We demonstrated the direct effect of GlADI on human TLR signaling. We predicted its mode of interaction with TLRs two and four by using the AlphaFold-predicted structure of GlADI and molecular docking. Furthermore, we showed that the immunomodulatory capacity of this virulent factor of Giardia depends on the maintenance of its 3D structure. Finally, we also showed the influence of this enzyme to exert specific responses on infant-like dendritic cells.

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Functional annotation of Trametes villosa genome was performed to search Class II peroxidase proteins in this white-rot fungus, which can be valuable for several biotechnological processes. After sequence identification and manual curation, five proteins were selected to build 3 D models by comparative modeling. Analysis of sequential and structural sequences from selected targets revealed the presence of two putative Lignin Peroxidase and three putative Manganese Peroxidase on this fungal genome. All 3 D models had a similar folding pattern from selected 3 D structure templates. After minimization and validation steps, the best 3 D models were subjected to docking studies and molecular dynamics to identify structural requirements and the interactions required for molecular recognition. Two reliable 3 D models of Class II peroxidases, with typical catalytic site and architecture, and its protein sequences are indicated to recombinant production in biotechnological applications, such as bioenergy.Communicated by Ramaswamy H. Sarma.

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The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.

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Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.

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Persisters are phenotypic variants of the bacterial population that survive against lethal doses of bactericidal antibiotics.These phenotypes are created in numerous bacterial species, including those of clinical significance, such as Salmonella Typhimurium. Since persister cells are associated with the failure of antibiotic treatment and infection recurrence, it is crucial to identify the mechanisms that influence the formation of these cells. The aim of this study is to investigate the persister cell formation and expression analysis as well as to predict the 3D structure of the genes involved in the production of persister cells. The presence of persisters in S. Typhimurium was determined by time dependent killing of different types of bactericidal antibiotics and expression of genes associated with persister cell formation which was assessed five hours after the addition of antibiotics by the qRT-PCR. Indeed, the 3D structural model of the proteins studied was predicted by performing several computational methods of retrieved primary protein sequences. The results of the study showed that the S. Typhimurium produced high levels of persister cells in the exposure of bactericidal antibiotics. Furthermore, qRT-PCR resulted in the fact that the expression of related genes was different depending on the type of antibiotic. Overall, this study provides information on the creation of persister cells and the role of different genes in the formation of these cells and structure of proteins involved in the production of persister cells in S. Typhimurium.

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To improve the availability of three-dimensional (3D) structures of HLA molecules, we created the pHLA3D database. In its first version, we modeled and published 106 3D structures of HLA class I molecules from the HLA-A, HLA-B, and HLA-C loci. This paper presents an update of this database, providing more 127 3D structures of HLA class II molecules (41 DR, 42 DQ, and 44 DP), predicted via homology modeling with MODELLER and SWISS-MODEL. These new 3D structures of HLA class II molecules are now freely available at pHLA3D (www.phla3d.com.br) for immunologists and other researchers working with HLA molecules.

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This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

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The understanding of the functional properties of mitochondrial transfer RNA (mt tRNAs) depend on the knowledge of its structure. tRNA acts as an interface between polynucleotides and polypeptides thus, they are key molecules in protein biosynthesis. The tRNA molecule has a functional design and, given its importance in the translation of mitochondrial genes, it is plausible that modifications of the structure can affect the synthesis of proteins and the functional properties of the mitochondria. In a previous work, the mitochondrial genome of an individual of the nesting Caretta caretta of the Colombian Caribbean was obtained, where specific mutations were identified in the only tRNALeu (CUN), tRNATrp and tRNALys genes. In order to analyze the effect of these mutations on these three mt tRNAs, the prediction of 2D and 3D structures was performed. Genes were sequenced in 11 nesting loggerhead turtles from the Colombian Caribbean. Two-dimensional structures were inferred using the ARWEN program, and three-dimensional structures were obtained with the RNA Composer 3D program. Two polymorphisms were identified in tRNATrp and another one was located in tRNALys, both specific to C. caretta. The thymine substitution in nucleotide position 14 of tRNATrp could constitute an endemic polymorphism of the nesting colony of the Colombian Caribbean. Two 2D and three 3D patterns were obtained for tRNATrp. In the case of tRNALys and tRNALeu 2D and 3D structures were obtained respectively, which showed compliance to canonical structures, with 4 bp in the D-arm, 4-5 bp in the T-arm, and 5 bp in the anticodon arm. Moderate deviations were found, such as a change in the number of nucleotides, elongation in loops or stems and non-Watson-Crick base pairing: adenine-adenine in stem D of tRNATrp, uracil-uracil and adenine-cytosine in the acceptor arm of the tRNALys and cytosine-cytosine in the anticodon stem of the tRNALeu. In addition, distortions or lack of typical interactions in 3D structures gave them unique characteristics. According to the size of the variable region (4-5 nt), the three analyzed tRNAs belong to class I. The interactions in the three studied tRNAs occur mainly between D loop-variable region, and between spacer bases-variable region, which classifies them as tRNA of typology II. The polymorphisms and structural changes described can, apparently, be post-transcriptionally stabilized. It will be crucial to perform studies at the population and functional levels to elucidate the synthetic pathways affected by these genes. This article analyses for the first time the 1D, 2D and 3D structures of the mitochondrial tRNALys, tRNATrp and tRNALeu in the loggerhead turtle.

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BACKGROUND: HPV16 infection is one of the main risk factors involved in the development of cervical cancer, mainly due to the high oncogenic potential of the viral proteins E6 and E7, which are involved in the different processes of malignant transformation. There is a broad spectrum of intratypical variation of E6, which is reflected in its high diversity, biological behavior, global distribution and risk of causing cervical cancer. Experimental studies have shown that the intratypical variants of the protein E6 from the European variants (E-G350, E-A176/G350, E-C188/G350) and Asian-American variants (AAa and AAc), are capable of inducing the differential expression of genes involved in the development of cervical cancer. RESULTS: An in silico analysis was performed to characterize the molecular effects of these variations using the structure of the HPV16 E6 oncoprotein (PDB: 4XR8; chain H) as a template. In particular, we evaluated the 3D structures of the intratypical variants by structural alignment, ERRAT, Ramachandran plots and prediction of protein disorder, which was further validated by molecular dynamics simulations. Our results, in general, showed no significant changes in the protein 3D structure. However, we observed subtle changes in protein physicochemical features and structural disorder in the N- and C-termini. CONCLUSIONS: Our results showed that mutations in the viral oncogene E6 of six high-risk HPV16 variants are effectively neutral and do not cause significant structural changes except slight variations of structural disorder. As structural disorder is involved in rewiring protein-protein interactions, these results suggest a differential pattern of interaction of E6 with the target protein P53 and possibly different patterns of tumor aggressiveness associated with certain types of variants of the E6 oncoprotein.

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DHHC palmitoyltransferases (DHHC-PATs) are very peculiar in that, outside the DHHC domain, they are very divergent even across orthologs from closely related species. This represents a challenge for the bioinformatic analyses of these proteins. Sequence-based analyses and predictions require a valid sequence alignment, which for this family of proteins requires extensive manual curation and this is difficult to attain for the nonspecialist. Here we present a simple method for the in silico analysis of the sequence of a particular PAT, that would allow for the identification of important structural features and functional residues in a PAT or PAT family.

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HLA epitope analysis emerged as a strategy to determine alloimmune risk in solid organ transplantation. However, it requires not only knowledge on HLA amino acids sequences, but also on HLA three-dimensional structures. Unfortunately, the number of structures available is still unsatisfactory. This work reports the modelling of 106 heterotrimeric (alpha chain + ß2M + peptide) HLA class I molecules. The models were generated by homology modelling using Modeller, refined using GalaxyRefine server, heterodimerized with Swiss-PDB Viewer and, finally, assessed as to their structural quality through Dali server. The final structures were made available through a free online database, pHLA3D (www.phla3d.com.br), developed in Ruby language using the Ruby on Rails web framework. Structural parameters were similar between refined molecules and their templates. The new database may improve HLA epitope analysis and better guide risk assessment in solid organ transplantation setting.

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A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short ß strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms.

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High-Level Structure (HLS) extraction in a set of images consists of recognizing 3D elements with useful information to the user or application. There are several approaches to HLS extraction. However, most of these approaches are based on processing two or more images captured from different camera views or on processing 3D data in the form of point clouds extracted from the camera images. In contrast and motivated by the extensive work developed for the problem of depth estimation in a single image, where parallax constraints are not required, in this work, we propose a novel methodology towards HLS extraction from a single image with promising results. For that, our method has four steps. First, we use a CNN to predict the depth for a single image. Second, we propose a region-wise analysis to refine depth estimates. Third, we introduce a graph analysis to segment the depth in semantic orientations aiming at identifying potential HLS. Finally, the depth sections are provided to a new CNN architecture that predicts HLS in the shape of cubes and rectangular parallelepipeds.

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Ribosomal RNA precursors undergo a series of structural and chemical modifications to generate matured RNA molecules that will comprise ribosomes. This maturation process involves a large set of accessory proteins as well as ribonucleases, responsible for removal of the external and internal transcribed spacers from the pre-rRNA. Early-diverging eukaryotes belonging to the Kinetoplastida class display several unique characteristics, in particular in terms of RNA synthesis and maturation. These peculiarities include the rRNA biogenesis and the extensive fragmentation of the large ribosomal subunit (LSU) rRNA. The role of specific endo- and exonucleases in the maturation of the unusual rRNA precursor of trypanosomatids remains largely unknown. One of the nucleases involved in rRNA processing is Rrp44, an exosome associated ribonuclease in yeast, which is involved in several metabolic RNA pathways. Here, we investigated the function of Trypanosoma brucei RRP44 orthologue (TbRRP44) in rRNA processing. Our results revealed that TbRRP44 depletion causes unusual polysome profile and accumulation of the complete LSU rRNA precursor, in addition to 5.8S maturation impairment. We also determined the crystal structure of TbRRP44 endonucleolytic domain. Structural comparison with Saccharomyces cerevisiae Rrp44 revealed differences in the catalytic site and substitutions of surface residues, which could provide molecular bases for the lack of interaction of RRP44 with the exosome complex in T. brucei.

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All living organisms need a DNA replication mechanism and it has been conserved in the three domains of life throughout evolutionary process. Primase is the enzyme responsible for synthesizing de novo RNA primers in DNA replication. Archaeo-Eukaryotic Primase (AEP) is the superfamily that typically forms a heterodimeric complex containing both a small catalytic subunit (PriS) and a large accessory noncatalytic subunit (PriL). Sulfolobus solfataricus is a model organism for research on the Genetics field. The aim of this work was to evaluate, via Bioinformatics tools, three mutations in the large subunit (PriL) of the archaeon Sulfolobus solfataricus. The aspartic acid residue in the positions (Asp) 62, (Asp) 235, (Asp) 241 have been substituted by glutamic acid (Glu). The highest positive free energy variation of the three substitutions analyzed occurred with the mutation at the (Asp) 241 site. The in silico analysis suggested that these mutations in PriL may destabilize its tridimensional structure interfering with replication mechanisms of Sulfolobus solfataricus. Moreover, it may also alter interactions with other molecules, making salt bridges, for instance.(AU)

Todos os organismos vivos necessitam de um eficiente mecanismo de replicação de DNA. Aolongo da evolução biológica foi observado que esse mecanismo é conservado nos três domínios da vida.Uma enzima importante que participa desse mecanismo é a RNA primase, a qual é responsável pela síntesede novo de iniciadores de RNA na replicação do DNA. Em Arquea-Eucariota, RNA Primase (AEP)tipicamente forma um complexo heterodimérico, que contém uma pequena subunidade catalítica (PriS) euma subunidade maior não catalítica acessória (PriL). Sulfolobus solfataricus é um organismo modelo deArquea para a pesquisa no campo da genética. O objetivo deste trabalho foi avaliar, por meio de ferramentasde bioinformática, três mutações pontuais na subunidade maior (PriL) de Sulfolobus solfataricus. Nassequências mutantes, os resíduos de ácido aspártico nas posições (Asp) 62, (Asp) 235, (Asp) 241 foramsubstituídos por ácido glutâmico (Glu). A maior variação de energia livre positiva das três mutaçõesanalisadas ocorreu no sítio (Asp) 241. A análise in silico sugeriu que essas mutações em PriL podemdesestabilizar sua estrutura tridimensional, interferindo com os mecanismos de replicação de Sulfolobussolfataricus. Além disso, podem alterar interações com outras moléculas, formando pontes salinas.(AU)

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All living organisms need a DNA replication mechanism and it has been conserved in the three domains of life throughout evolutionary process. Primase is the enzyme responsible for synthesizing de novo RNA primers in DNA replication. Archaeo-Eukaryotic Primase (AEP) is the superfamily that typically forms a heterodimeric complex containing both a small catalytic subunit (PriS) and a large accessory noncatalytic subunit (PriL). Sulfolobus solfataricus is a model organism for research on the Genetics field. The aim of this work was to evaluate, via Bioinformatics tools, three mutations in the large subunit (PriL) of the archaeon Sulfolobus solfataricus. The aspartic acid residue in the positions (Asp) 62, (Asp) 235, (Asp) 241 have been substituted by glutamic acid (Glu). The highest positive free energy variation of the three substitutions analyzed occurred with the mutation at the (Asp) 241 site. The in silico analysis suggested that these mutations in PriL may destabilize its tridimensional structure interfering with replication mechanisms of Sulfolobus solfataricus. Moreover, it may also alter interactions with other molecules, making salt bridges, for instance.

Todos os organismos vivos necessitam de um eficiente mecanismo de replicação de DNA. Ao longo da evolução biológica foi observado que esse mecanismo é conservado nos três domínios da vida. Uma enzima importante que participa desse mecanismo é a RNA primase, a qual é responsável pela síntese de novo de iniciadores de RNA na replicação do DNA. Em Arquea-Eucariota, RNA Primase (AEP) tipicamente forma um complexo heterodimérico, que contém uma pequena subunidade catalítica (PriS) e uma subunidade maior não catalítica acessória (PriL). Sulfolobus solfataricus é um organismo modelo de Arquea para a pesquisa no campo da genética. O objetivo deste trabalho foi avaliar, por meio de ferramentas de bioinformática, três mutações pontuais na subunidade maior (PriL) de Sulfolobus solfataricus. Nas sequências mutantes, os resíduos de ácido aspártico nas posições (Asp) 62, (Asp) 235, (Asp) 241 foram substituídos por ácido glutâmico (Glu). A maior variação de energia livre positiva das três mutações analisadas ocorreu no sítio (Asp) 241. A análise in silico sugeriu que essas mutações em PriL podem desestabilizar sua estrutura tridimensional, interferindo com os mecanismos de replicação de Sulfolobus solfataricus. Além disso, podem alterar interações com outras moléculas, formando pontes salinas.

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GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

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Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular mass of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose. It had the ability to agglutinate all types of human erythrocytes, as well as rabbit red blood cells. Circular dichroism analyzes have indicated that this lectin possessed an α/ß fold with a predominance of ß structures. This was consistent with the structure of the protein that was determined by the X-ray diffraction techniques. MCL was crystallized in the space group C21 and it diffracted to 1.79 Å resolution. Two monomers were found in the asymmetric unit and they formed dimers in solution. The protein has shown to be a member of the ß-trefoil family, with three sugar binding sites per monomer. In accord with fluorescence-based thermal shift assays, we observed that the MCL Tm increased about 10 °C in the presence of galactose. Furthermore, we have determined the complete amino acid sequence by cDNA sequencing. The gene had two ORF2 proteins, one resulting in a 180 residue protein with a theoretical molecular mass of 20227 Da, and another resulting in a 150 residue protein with a theoretical molecular mass of 16911 Da. The difference between the theoretical and experimental values was due to the presence of a glycosylation that was observed by the glycosylation assay. A positive microbial agglutination and a growth inhibition activity were observed against Gram-negative and Gram-positive bacteria. The M. californianus lectin is the fourth member of the recently proposed new family of lectins that have been reported to date, occurring only in mollusks belonging to the family Mytilidae. It is the first member to be glycosylated and with a strong tendency to form large oligomers.

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