Jonathan L. McMurry

Analysis of the Cytoplasmic Domains of Salmonella FlhA and Interactions with Components of the Flagellar Export Machinery

Most flagellar proteins are exported via a type III export apparatus which, in part, consists of the membrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR and is housed within the membrane-supramembrane ring formed by FliF subunits. Salmonella FlhA is a 692-residue integral membrane protein with eight predicted transmembrane spans. Its function is not understood, but it is necessary for flagellar export. We have created mutants in which potentially important sequences were deleted. FlhA lacking the amino-terminal sequence prior to the first transmembrane span failed to complement and was dominant negative, suggesting that the sequence is required for function. Similar effects were seen in a variant lacking a highly conserved domain (FHIPEP) within a putative cytoplasmic loop. Scanning deletion analysis of the cytoplasmic domain (FlhAc) demonstrated that substantially all of FlhAc is required for efficient function. Affinity blotting showed that FlhA interacts with several other export apparatus membrane proteins. The implications of these findings are discussed, and a model of FlhA within the export apparatus is presented.

Cannabinoid receptor-G protein interactions: Gαi1-bound structures of IC3 and a mutant with altered G protein specificity

The structure of the C‐terminal region of the third cytoplasmic loop (IC3) of the cannabinoid receptor one (CB1) bound to Gαi1 has been determined using transferred nuclear Overhauser effects (NOEs). The wild‐type IC3 sequence is helical when associated with Gαi1. In contrast, a peptide containing the amino‐acid inversion, Ala341‐Leu342 adopts a single turn. These findings correlate with the attenuated Gi association of CB1 with the Ala341‐Leu342 mutation previously observed in vivo and the diminished stimulation of Gαi1 GTPase activity by the corresponding peptide demonstrated in vitro here. These results, the first to report the structure of a GPCR domain while associated with G protein, imply the C‐terminus of CB1 IC3, a region with high‐sequence conservation among G‐protein coupled receptors, must be helical for efficient coupling and activation of the Gi protein.

Analysis of an Engineered Salmonella Flagellar Fusion Protein, FliR-FlhB

Salmonella FliR and FlhB are membrane proteins necessary for flagellar export. In Clostridium a fliR-flhB fusion gene exists. We constructed a similar Salmonella fusion gene which is able to complement fliR, flhB, and fliR flhB null strains. Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB proteins cleavage properties. We conclude that the FliR and FlhB proteins are physically associated in the wild-type Salmonella basal body, probably in a 1:1 ratio.

Flagellar Formation in C-Ring-Defective Mutants by Overproduction of FliI, the ATPase Specific for Flagellar Type III Secretion

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a Fla(-) phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.

Characterization of Myxococcus xanthus MazF and implications for a new point of regulation

During development, Myxococcus xanthus cells undergo programmed cell death (PCD) whereby 80% of vegetative cells die. Previously, the MazF RNA interferase has been implicated in this role. Recently, it was shown that deletion of the mazF gene does not eliminate PCD in wild‐type strain DK1622 as originally seen in DZF1. To clarify the role of MazF, recombinant enzyme was characterized using a highly sensitive assay in the presence and absence of the proposed antitoxin MrpC. In contrast to previous reports that MrpC inhibits MazF activity, the hydrolysis rate was enhanced in a concentration‐dependent manner with MrpC or MrpC2, an N‐terminally truncated form of MrpC. Furthermore, MazF transcripts were not detected until 6–8 h post‐induction, suggesting an antitoxin is unnecessary earlier. Potential MazF targets were identified and their transcript levels were shown to decline in DK1622 while remaining steady in a mazF deletion strain. Elimination of the mazF hydrolysis site in the nla6 transcript resulted in overproduction of the mRNA. Thus, MazF negatively regulates specific transcripts. Additionally, we show that discrepancies in the developmental phenotypes caused by removal of mazF in DK1622 and DZF1 are due to the presence of the pilQ1 allele in the latter strain.

Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch.

The bacterial flagellum is a complex macromolecular machine consisting of more than 20 000 proteins, most of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point in flagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rod and hook proteins to filament ones. How the switch works is a subject of intense interest. FliK and FlhB play central roles. In the present study, two optical biosensing methods were used to characterize FliK-FlhB interactions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Binding was found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type and variant FlhBs had similar kinetic profiles and apparent affinities, which ranged between 1 and 10.5 microM, suggesting that the specificity switch is more complex than presently understood. Other binding experiments provided evidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) and NMR experiments were performed to identify a cyclic intermediate product whose existence supports the mechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalytic cleavage is necessary for proper substrate specificity switching, it does not result in an altered interaction with FliK, strongly suggesting the involvement of other proteins in the mechanism.

The Helicobacter pylori Anti-Sigma Factor FlgM Is Predominantly Cytoplasmic and Cooperates with the Flagellar Basal Body Protein FlhA

Helicobacter pylori requires flagellar motility and orientation to persist actively in its habitat. A particular feature of flagella in most Helicobacter species including H. pylori is a membraneous flagellar sheath. The anti-sigma factor FlgM of H. pylori is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g., FlgM of Salmonella spp., whose regulatory function is intimately coupled to its secretion through the flagellar type III secretion system. The aim of the present study was to characterize the localization and secretion of the short H. pylori FlgM in the presence of a flagellar sheath and to elucidate its interaction with other flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate with FlgM for regulation. H. pylori FlgM was only released into the medium in minor amounts in wild-type bacteria, where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellar fraction. FlgM was expressed in flhA mutants and was less soluble and differentially localized in bacterial fractions of the flhA mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5 translational fusions were generated and expressed in H. pylori. FlgM displayed a predominantly polar distribution and interacted with the C-terminal domain of FlhA (FlhA(C)). We suggest that, in H. pylori, FlgM secretion may not be paramount for its regulatory function and that protein interactions at the flagellar basal body may determine the turnover and localization of functional FlgM.

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin.

Using interferometry‐based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation‐independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM‐binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM‐binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca2+ binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca2+ dependence of CaM binding. Hence, changes in the Ca2+ sensitivity of CaM binding always imply changes in the NOS–CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca2+ sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.

Optical biosensing: Kinetics of protein A‐IGG binding using biolayer interferometry

An undergraduate biochemistry laboratory experiment has been developed using biolayer interferometry (BLI), an optical biosensing technique similar to surface plasmon resonance (SPR), in which students obtain and analyze kinetic data for a protein‐protein interaction. Optical biosensing is a technique of choice to determine kinetic and affinity constants for biomolecular interactions. Measurements can be made in real‐time without labels, making biosensing particularly appropriate for the teaching laboratory. In the described exercise, students investigate the kinetics of Protein A‐human Immunoglobin G binding under conditions that mimic simple 1:1 binding. Students prepare appropriate serial dilutions of IgG and set up a microplate for the experiment by aliquotting biotinylated Protein A, buffer, and IgG solutions. A commercial BLI sensor, the FortéBio Octet QK, is used to measure binding. While data are collected students prepare a spreadsheet with which they will simulate the data to determine kon, koff, and KD. Raw data from the sensor are then exported to the spreadsheets for analysis. Optimized experiment timing, regeneration methods and other parameters are described to increase throughput and reduce cost. The experiment is readily adaptable to other biosensing platforms such as SPR instruments. Biochemistry and Molecular Biology Education Vol. 38, No. 6, pp. 400‐407, 2010

Helicobacter Pylori Hydrogenase Accessory Protein HypA and Urease Accessory Protein UreG Compete with Each Other for UreE Recognition

BACKGROUND The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni(2+) into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH. METHODS Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG. RESULTS Using both methods, affinity constants were found to be 5nM and 13nM for HypA-UreE and 8μM and 14μM for UreG-UreE. Neither Zn(2+) nor Ni(2+) had an effect on the kinetics or stability of the HypA-UreE complex. By contrast, addition of Zn(2+), but not Ni(2+), altered the kinetics and greatly increased the stability of the UreE-UreG complex, likely due in part to Zn(2+)-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition. GENERAL SIGNIFICANCE Factors influencing the pathogens nickel budget are important to understand pathogenesis and for future drug design.