Variations of the NodB Architecture Are Attuned to Functional Specificities into and beyond the Carbohydrate Esterase Family 4.

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families.

Human Aldehyde Dehydrogenases: A Superfamily of Similar Yet Different Proteins Highly Related to Cancer.

The superfamily of human aldehyde dehydrogenases (hALDHs) consists of 19 isoenzymes which are critical for several physiological and biosynthetic processes and play a major role in the organism's detoxification via the NAD(P) dependent oxidation of numerous endogenous and exogenous aldehyde substrates to their corresponding carboxylic acids. Over the last decades, ALDHs have been the subject of several studies as it was revealed that their differential expression patterns in various cancer types are associated either with carcinogenesis or promotion of cell survival. Here, we attempt to provide a thorough review of hALDHs' diverse functions and 3D structures with particular emphasis on their role in cancer pathology and resistance to chemotherapy. We are especially interested in findings regarding the association of structural features and their changes with effects on enzymes' functionalities. Moreover, we provide an updated outline of the hALDHs inhibitors utilized in experimental or clinical settings for cancer therapy. Overall, this review aims to provide a better understanding of the impact of ALDHs in cancer pathology and therapy from a structural perspective.

Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA.

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.

Identification of a peptide ligand for human ALDH3A1 through peptide phage display: Prediction and characterization of protein interaction sites and inhibition of ALDH3A1 enzymatic activity.

Aldehyde dehydrogenase 3A1 (ALDH3A1) by oxidizing medium chain aldehydes to their corresponding carboxylic acids, is involved in the detoxification of toxic byproducts and is considered to play an important role in antioxidant cellular defense. ALDH3A1 has been implicated in various other functions such as cell proliferation, cell cycle regulation, and DNA damage response. Recently, it has been identified as a putative biomarker of prostate, gastric, and lung cancer stem cell phenotype. Although ALDH3A1 has multifaceted functions in both normal and cancer homeostasis, its modes of action are currently unknown. To this end, we utilized a random 12-mer peptide phage display library to identify efficiently human ALDH3A1-interacting peptides. One prevailing peptide (P1) was systematically demonstrated to interact with the protein of interest, which was further validated in vitro by peptide ELISA. Bioinformatic analysis indicated two putative P1 binding sites on the protein surface implying biomedical potential and potent inhibitory activity of the P1 peptide on hALDH3A1 activity was demonstrated by enzymatic studies. Furthermore, in search of potential hALDH3A1 interacting players, a BLASTp search demonstrated that no protein in the database includes the full-length amino acid sequence of P1, but identified a list of proteins containing parts of the P1 sequence, which may prove potential hALDH3A1 interacting partners. Among them, Protein Kinase C Binding Protein 1 and General Transcription Factor II-I are candidates of high interest due to their cellular localization and function. To conclude, this study identifies a novel peptide with potential biomedical applications and further suggests a list of protein candidates be explored as possible hALDH3A1-interacting partners in future studies.

Structural Evolution of Primate Glutamate Dehydrogenase 2 as Revealed by In Silico Predictions and Experimentally Determined Structures.

Glutamate dehydrogenase (GDH) interconverts glutamate to a-ketoglutarate and ammonia, interconnecting amino acid and carbohydrate metabolism. In humans, two functional GDH genes, GLUD1 and GLUD2, encode for hGDH1 and hGDH2, respectively. GLUD2 evolved from retrotransposition of the GLUD1 gene in the common ancestor of modern apes. These two isoenzymes are involved in the pathophysiology of human metabolic, neoplastic, and neurodegenerative disorders. The 3D structures of hGDH1 and hGDH2 have been experimentally determined; however, no information is available about the path of GDH2 structure changes during primate evolution. Here, we compare the structures predicted by the AlphaFold Colab method for the GDH2 enzyme of modern apes and their extinct primate ancestors. Also, we analyze the individual effect of amino acid substitutions emerging during primate evolution. Our most important finding is that the predicted structure of GDH2 in the common ancestor of apes was the steppingstone for the structural evolution of primate GDH2s. Two changes with a strong functional impact occurring at the first evolutionary step, Arg443Ser and Gly456Ala, had a destabilizing and stabilizing effect, respectively, making this step the most important one. Subsequently, GDH2 underwent additional modifications that fine-tuned its enzymatic properties to adapt to the functional needs of modern-day primate tissues.

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles.

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell's metabolism for the benefit of the bacterium to bypassing the host's defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology.

INTRODUCTION: Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9 Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics.

Catalytic activity regulation through post-translational modification: the expanding universe of protein diversity.

Protein composition is restricted by the genetic code to a relatively small number of natural amino acids. Similarly, the known three-dimensional structures adopt a limited number of protein folds. However, proteins exert a large variety of functions and show a remarkable ability for regulation and immediate response to intracellular and extracellular stimuli. To some degree, the wide variability of protein function can be attributed to the post-translational modifications. Post-translational modifications have been observed in all kingdoms of life and give to proteins a significant degree of chemical and consequently functional and structural diversity. Their importance is partly reflected in the large number of genes dedicated to their regulation. So far, hundreds of post-translational modifications have been observed while it is believed that many more are to be discovered along with the technological advances in sequencing, proteomics, mass spectrometry and structural biology. Indeed, the number of studies which report novel post translational modifications is getting larger supporting the notion that their space is still largely unexplored. In this review we explore the impact of post-translational modifications on protein structure and function with emphasis on catalytic activity regulation. We present examples of proteins and protein families whose catalytic activity is substantially affected by the presence of post translational modifications and we describe the molecular basis which underlies the regulation of the protein function through these modifications. When available, we also summarize the current state of knowledge on the mechanisms which introduce these modifications to protein sites.

Cryo-EM density map fitting driven in-silico structure of human soluble guanylate cyclase (hsGC) reveals functional aspects of inter-domain cross talk upon NO binding.

The human soluble Guanylate Cyclase (hsGC) is a heterodimeric heme-containing enzyme which regulates many important physiological processes. In eukaryotes, hsGC is the only known receptor for nitric oxide (NO) signaling. Improper NO signaling results in various disease conditions such as neurodegeneration, hypertension, stroke and erectile dysfunction. To understand the mechanisms of these diseases, structure determination of the hsGC dimer complex is crucial. However, so far all the attempts for the experimental structure determination of the protein were unsuccessful. The current study explores the possibility to model the quaternary structure of hsGC using a hybrid approach that combines state-of-the-art protein structure prediction tools with cryo-EM experimental data. The resultant 3D model shows close consistency with structural and functional insights extracted from biochemistry experiment data. Overall, the atomic-level complex structure determination of hsGC helps to unveil the inter-domain communication upon NO binding, which should be of important usefulness for elucidating the biological function of this important enzyme and for developing new treatments against the hsGC associated human diseases.

Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation.

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.

Maturation of 6S regulatory RNA to a highly elongated structure.

As bacterial populations leave the exponential growth phase and enter the stationary phase, their patterns of gene expression undergo marked changes. A key effector of this change is 6S RNA, which is a highly conserved regulatory RNA that impedes the transcription of genes associated with exponential growth by forming an inactivating ternary complex with RNA polymerase and sigma factor σ(70) (σ(70)-RNAP). In Escherichia coli, the endoribonuclease RNase E generates 6S RNA by specific cleavage of a precursor that is nearly twice the size of the 58 kDa mature form. We have explored recognition of the precursor by RNase E, and observed that processing is inhibited under conditions of excess substrate. This finding supports a largely distributive mechanism, meaning that each round of catalysis results in enzyme dissociation and re-binding to the substrate. We show that the precursor molecule and the mature 6S share a structural core dominated by an A-type helix, indicating that processing is not accompanied by extensive refolding. Both precursor and mature forms of 6S have a highly stable secondary structure, adopt an elongated shape, and show the potential to form dimers under specific conditions; nonetheless, 6S has a high structural plasticity that probably enables it to be structurally adapted upon binding to its cognate protein partners. Analysis of the 6S-σ(70)-RNAP complex by native mass spectrometry reveals a stable association with a stoichiometry of 1:1:1. A theoretical 3D model of mature 6S is presented, which is consistent with the experimental data and supports a previously proposed structure with a small stem-loop inside the central bubble.

Electroelution of nucleic acids from polyacrylamide gels: a custom-made, agarose-based electroeluter.

Polyacrylamide electrophoresis is routinely used for small-scale preparative and analytical separations. The incomparably high-resolution separations achieved by this technique, however, have not been widely exploited to the large-scale preparative isolation of biological molecules from contaminants, mainly because of difficulties in the recovery of the desired molecule from the gel matrix. Electroelution is an effective procedure applied for this purpose. However, commercially available, high-cost electroeluters are required for achieving high recovery yields. Here, we describe a custom-made electroeluter that combines low-cost, high-recovery yields, short times of electroelution, and convenience in the manipulation of sensitive samples.

Structure determination through homology modelling and torsion-angle simulated annealing: application to a polysaccharide deacetylase from Bacillus cereus.

The structure of BC0361, a polysaccharide deacetylase from Bacillus cereus, has been determined using an unconventional molecular-replacement procedure. Tens of putative models of the C-terminal domain of the protein were constructed using a multitude of homology-modelling algorithms, and these were tested for the presence of signal in molecular-replacement calculations. Of these, only the model calculated by the SAM-T08 server gave a consistent and convincing solution, but the resulting model was too inaccurate to allow phase determination to proceed to completion. The application of slow-cooling torsion-angle simulated annealing (started from a very high temperature) drastically improved this initial model to the point of allowing phasing through cycles of model building and refinement to be initiated. The structure of the protein is presented with emphasis on the presence of a C(α)-modified proline at its active site, which was modelled as an α-hydroxy-L-proline.

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains.

BACKGROUND: The central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs. RESULTS: Based on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments. CONCLUSIONS: The relatedness of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome.

LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). Only a few members of this family have been biochemically characterized including N-acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins), N,N'-diacetylchitobiose (GlcNAc(2)) and lipoglycopeptide antibiotic de-N-acetylases. All these enzymes share a common feature in that they de-N-acetylate the N-acetyl-D-glucosamine (GlcNAc) moiety of their substrates. The bc1534 gene has previously been cloned and expressed in Escherichia coli. The recombinant enzyme was purified and its 3D structure determined. In this study, the bc3461 gene from B. cereus ATCC14579 was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC 3.5.1.-) and BC3461 were biochemically characterized. The enzymes have different molecular masses, pH and temperature optima and broad substrate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers (GlcNAc(2), GlcNAc(3) and GlcNAc(4)), as well as GlcNAc-1P, N-acetyl-D-glucosamine-1 phosphate; GlcNAc-6P, N-acetyl-D-glucosamine-6 phosphate; GalNAc, N-acetyl-D-galactosamine; ManNAc, N-acetyl-D-mannosamine; UDP-GlcNAc, uridine 5'-diphosphate N-acetyl-D-glucosamine. However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B. cereus, N-acetyl-D-glucosaminyl-(beta-1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) or N-acetyl-D-GlcN-Nalpha1-6-D-myo-inositol-1-HPO(4)-octadecyl (GlcNAc-I-P-C(18)). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc(2) revealed that GlcNAc(2) is the favored substrate for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key residues, highlighting their importance for the catalytic mechanism and the substrate specificity of the enzyme. The catalytic efficiencies of BC1534 variants were significantly decreased compared to the native enzyme. An alignment-based tree places both de-N-acetylases in functional categories that are different from those of other LmbE proteins.

On the quaternary association of the type III secretion system HrcQB-C protein: experimental evidence differentiates among the various oligomerization models.

The HrcQB protein from the plant pathogen Pseudomonas syringae is a core component of the bacterial type III secretion apparatus. The core consists of nine proteins widely conserved among animal and plant pathogens which also share sequence and structural similarities with proteins from the bacterial flagellum. Previous studies of the carboxy-terminal domain of HrcQB (HrcQB-C) and its flagellar homologue, FliN-C, have revealed extensive sequence and structural homologies, similar subcellular localization, and participation in analogous protein-protein interaction networks. It is not clear however whether the similarities between the two proteins extend to the level of quaternary association which is essential for the formation of higher-order structures within the TTSS. Even though the crystal structure of the FliN is a dimer, more detailed studies support a tetrameric donut-like association. However, both models, dimer and donut-like tetramer, are quite different from the crystallographic elongated dimer of dimers of the HrcQB-C. To resolve this discrepancy we performed a multidisciplinary investigation of the quaternary association of the HrcQB-C, including mass-spectrometry, electrophoresis in non-reductive conditions, gel filtration, glutaraldehyde cross-linking and small angle X-ray scattering. Our experiments indicate that stable tetramers of elongated shape are assembled in solution, in agreement with the results of crystallographic studies. Circular dichroism data are consistent with a dimer-dimer interface analogous to the one established in the crystal structure. Finally, molecular dynamics simulations reveal the relative orientation of the dimers forming the tetramers and the possible differences from that of the crystal structure.

Molecular Dynamics Simulations of BcZBP, A Deacetylase from Bacillus cereus: Active Site Loops Determine Substrate Accessibility and Specificity.

BcZBP is an LmbE-like, homohexameric, zinc-dependent deacetylase from the opportunistic pathogen Bacillus cereus with three, thus far uncharacterized, homologues in B. anthracis. Although its specific substrate is still unknown, the enzyme has been shown to preferentially deacetylate N-acetylglucosamine and diacetylchitobiose via an active site based on a zinc-binding motif of the type HXDDXnH. In the crystal structure, the active site is located at a deep and partially blocked cleft formed at the interface between monomers related by the molecular 3-fold axis, although the major, in structural terms, building block of the enzyme is not the trimer, but the intertwined dimer. Here, we report results from a 50 ns molecular dynamics simulation of BcZBP in explicit solvent with full electrostatics and show that (i) the view of the intertwined dimer as the major structural and functional building block of this class of hexameric enzymes is possibly an oversimplification of the rather complex dynamics observed in the simulation, (ii) the most mobile (with respect to their atomic fluctuations) parts of the structure coincide with three surface loops surrounding the active site, and (iii) these mobile loops define the active site's accessibility, and may be implicated in the determination of the enzyme's specificity.

Determination of protein oligomerization state: two approaches based on glutaraldehyde crosslinking.

Many biochemical and biophysical methods can be used to characterize the oligomerization state of proteins. One of the most widely used is glutaraldehyde crosslinking, mainly because of the minimum equipment and reagents required. However, the crosslinking procedures currently in use are impaired by the low specificity of the reagent, which can chemically bond any two amino groups that are close in space. Thus, extensive and time-consuming investigation of the reaction conditions is usually required. Here we describe two approaches based on glutaraldehyde that readily give reliable results.

Crystal structure of the BcZBP, a zinc-binding protein from Bacillus cereus.

Bacillus cereus is an opportunistic pathogenic bacterium closely related to Bacillus anthracis, the causative agent of anthrax in mammals. A significant portion of the B. cereus chromosomal genes are common to B. anthracis, including genes which in B. anthracis code for putative virulence and surface proteins. B. cereus thus provides a convenient model organism for studying proteins potentially associated with the pathogenicity of the highly infectious B. anthracis. The zinc-binding protein of B. cereus, BcZBP, is encoded from the bc1534 gene which has three homologues to B. anthracis. The protein exhibits deacetylase activity with the N-acetyl moiety of the N-acetylglucosamine and the diacetylchitobiose and triacetylchitotriose. However, neither the specific substrate of the BcZBP nor the biochemical pathway have been conclusively identified. Here, we present the crystal structure of BcZBP at 1.8 A resolution. The N-terminal part of the 234 amino acid protein adopts a Rossmann fold whereas the C-terminal part consists of two beta-strands and two alpha-helices. In the crystal, the protein forms a compact hexamer, in agreement with solution data. A zinc binding site and a potential active site have been identified in each monomer. These sites have extensive similarities to those found in two known zinc-dependent hydrolases with deacetylase activity, MshB and LpxC, despite a low degree of amino acid sequence identity. The functional implications and a possible catalytic mechanism are discussed.

Purification, crystallization and preliminary characterization of a putative LmbE-like deacetylase from Bacillus cereus.

The Bacillus cereus BC1534 protein, a putative deacetylase from the LmbE family, has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Crystals of the 26 kDa protein grown from MPD and acetate buffer belong to space group R32, with unit-cell parameters a = b = 76.7, c = 410.5 A (in the hexagonal setting). A complete native data set was collected to a resolution of 2.5 A from a single cryoprotected crystal using synchrotron radiation. As BC1534 shows significant sequence homology with an LmbE-like protein of known structure from Thermus thermophilus, molecular replacement will be used for crystal structure determination.