A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183–383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine

Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

ABSTRACTRhamnose catabolism inRhizobium leguminosarumwas found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded byrhaSTPQand on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis ofrhaKshowed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the allelesrhaK72andrhaK73were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined byrhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either therhaK72orrhaK73allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly.IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are thecarbohydrateuptaketransporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport.

Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 fromMethanopyrus kandleri

Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such asMethanopyrus kandlericontain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 fromM. kandleriin apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified.