Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study.

Domains are the three-dimensional building blocks of proteins. An individual domain can occur in a variety of domain architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino acid sequences of a single domain across architectural contexts. The ability to distinguish different evolutionary outcomes of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein, lead to insights and hypotheses about function, and guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We analyzed 16 domain architectures that included 94% of all CheW-like domains found in nature. We identified six Classes of CheW-like domains with presumed functional differences. CheV and most CheW proteins contained Class 1 domains, whereas some CheW proteins contained Class 6 (~20%) or Class 2 (~1%) domains instead. Most CheA proteins contained Class 3 domains. CheA proteins with multiple Hpt domains contained Class 4 domains. CheA proteins with two CheW-like domains contained one Class 3 and one Class 5. We also created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available.

Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1.

Two-component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high-osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co-crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co-crystal structure of Ypd1 and a near wild-type variant of the receiver domain of Ssk1 (Ssk1-R2-W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1-receiver domain complexes, biochemical determination of binding affinities for Ssk1-R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/Ssk1-R2-W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.

Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD.

The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, ß = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.

Secondary metabolites produced by fungi derived from a microbial mat encountered in an iron-rich natural spring.

A collection of fungal isolates was obtained from a complex microbial mat, which occupied an iron-rich freshwater spring that feeds into Clear Creek, Golden, Colorado, USA. Two of the fungal isolates, a Glomeromycete (possible Entrophospora sp.) and a Dothideomycete (possible Phaeosphaeria sp.), were investigated for bioactive secondary metabolites. In total, six new compounds consisting of clearanols A-E (5, 6, 10-12) and disulochrin (7) were purified and their structures were determined. Disulochrin exhibited modest antibacterial activity against methicillin-resistant Staphylococcus aureus, whereas clearanol C showed weak inhibitory activity against Candida albicans biofilm formation.