Automated identification of chalcogen bonds in AlphaFold protein structure database files: is it possible?

Protein structure prediction and structural biology have entered a new era with an artificial intelligence-based approach encoded in the AlphaFold2 and the analogous RoseTTAfold methods. More than 200 million structures have been predicted by AlphaFold2 from their primary sequences and the models as well as the approach itself have naturally been examined from different points of view by experimentalists and bioinformaticians. Here, we assessed the degree to which these computational models can provide information on subtle structural details with potential implications for diverse applications in protein engineering and chemical biology and focused the attention on chalcogen bonds formed by disulphide bridges. We found that only 43% of the chalcogen bonds observed in the experimental structures are present in the computational models, suggesting that the accuracy of the computational models is, in the majority of the cases, insufficient to allow the detection of chalcogen bonds, according to the usual stereochemical criteria. High-resolution experimentally derived structures are therefore still necessary when the structure must be investigated in depth based on fine structural aspects

Naked Metal Cations Swimming in Protein Crystals

The presence of isolated metal cations, far from any other atom, is not uncommon in protein crystal structures. A systematic survey of the Protein Data Bank showed that nearly 8% of the metal cations are naked, more frequently if they can interact only electrostatically with their neighbors. Surprisingly, this seemed to be only weakly related to the crystallographic resolution

The G-M-N motif determines ion selectivity in the yeast magnesium channel Mrs2p

The highly conserved G-M-N motif of the CorA-Mrs2-Alr1 family of Mg2+ channels has been shown to be essential for Mg2+ transport. We performed random mutagenesis of the G-M-N sequence of Saccharomyces cerevisiae Mrs2p in an unbiased genetic screen. A large number of mutants still capable of Mg2+ influx, albeit below the wild-type level, were generated. Growth complementation assays, performed in media supplemented with Ca2+ or Co2+ or Mn2+ or Zn2+ at varying concentrations, lead to identification of mutants with reduced growth in the presence of Mn2+ and Zn2+. We hereby conclude that (1) at least two, but predominantly all three amino acids of the G-M-N motif must be replaced by certain combinations of other amino acids to remain functional, (2) replacement of any single amino acid within the G-M-N motif always impairs the function of Mrs2p, and (3) we show that the G-M-N motif determines ion selectivity, likely in concurrence with the negatively charged loop at the entrance of the channel thereby forming the Mrs2p selectivity filter

Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete <it>Glomerella cingulata </it>is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by <it>Glomerella </it>(<it>anamorph Colletotrichum) spp</it>.</p> <p>Results</p> <p>Heterologous expression of active <it>G. cingulata </it>GDH has been achieved in both <it>Escherichia coli </it>and <it>Pichia pastoris</it>, however, the expressed volumetric activity was about 4800-fold higher in <it>P. pastoris</it>. Expression in <it>E. coli </it>resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a <it>P. pastoris </it>transformant resulted in an expression of 48,000 U L^-1of GDH activity (57 mg L^-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in <it>P. pastoris </it>and the wild-type enzyme from its natural fungal source.</p> <p>Conclusions</p> <p>The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in <it>P. pastoris </it>facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.</p

From chlorite dismutase towards HemQ -the role of the proximal H-bonding network in haeme binding

Synopsis Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from &apos;Candidatus Nitrospira defluvii&apos; (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV-vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure-function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria

The Structure and Regulation of Human Muscle α-Actinin

SummaryThe spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins

The sarcomeric cytoskeleton:From molecules to motion

ABSTRACT Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise.</jats:p