Loma Linda University

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Mark Johnson, PhD
Assoc Res Prof, Basic Sciences
School of Medicine
Member, Microbiology&Molecular Gen, SM, Faculty of Graduate Studies
Publications    Scholarly Journals--Published
  • Watts, K. J., M. S. Johnson & B. L. Taylor, "Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer", J Bacteriol 190: 2118-2127, 2008.  doi: 10.1128/JB.01858-07   ( 1/2008 - 12/2008 ) Link...
    Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two alpha-helices separated by a structured loop. The proximal signaling domain consisted of two alpha-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the beta-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.
  • Amin, D. N., B. L. Taylor, and M. S. Johnson.  "Organization of the aerotaxis receptor aer in the membrane of Escherichia coli". J Bacteriol 189:7206-12  (2007). doi: 10.1128/JB.00871-07 ( 8/2007 ) Link...
    The Aer receptor guides Escherichia coli to specific oxygen and energy-generating niches. The input sensor in Aer is an FAD-binding, PAS domain, which is separated from a HAMP/signaling output domain by two membrane spanning segments that flank a short (four amino acid) periplasmic loop. In this study, we determined the overall membrane organization of Aer by introducing combinations of residues that allowed us to differentiate intra- from inter-dimeric collisions. Collisions between proximal residues in the membrane anchor were exclusively intra- or inter-dimeric, but, with one exception, not both. Crosslinking profiles were consistent with a rigid, rather than flexible periplasmic loop, and a tilted TM2 helix that crossed TM2'''' at residue V197C, near the center of the lipid bilayer. The periplasmic loop formed a stable neighborhood that i) included a maximum of three Aer dimers, ii) did not swap neighbors over time and iii) appeared to be constrained by interactions in the cytosolic signaling domain.
  • Taylor, B. L., K. J. Watts, M. S. Johnson. "Oxygen and Redox Sensing by Two-Component Systems That Regulate Behavioral Responses: Behavioral Assays and Structural Studies of Aer Using In Vivo Disulfide Cross-Linking." Methods in Enzymology 422. (2007): 190-232. ( 7/2007 ) Link...
    A remarkable increase in the number of annotated aerotaxis (oxygen-seeking) and redox taxis sensors can be attributed to recent advances in bacterial genomics. However, in silico predictions should be supported by behavioral assays and genetic analyses that confirm an aerotaxis or redox taxis function. This chapter presents a collection of procedures that have been highly successful in characterizing aerotaxis and redox taxis in Escherichia coli. The methods are described in enough detail to enable investigators of other species to adapt the procedures for their use. A gas flow cell is used to quantitate the temporal responses of bacteria to a step increase or decrease in oxygen partial pressure or redox potential. Bacterial behavior in spatial gradients is analyzed using optically flat capillaries and soft agar plates (succinate agar or tryptone agar). We describe two approaches to estimate the preferred partial pressure of oxygen that attracts a bacterial species; this concentration is important for understanding microbial ecology. At the molecular level, we describe procedures used to determine the structure and topology of Aer, a membrane receptor for aerotaxis. Cysteine-scanning mutagenesis and in vivo disulfide cross-linking procedures utilize the oxidant Cu(II)-(1,10-phenanthroline)3 and bifunctional sulfhydryl-reactive probes. Finally, we describe methods used to determine the boundaries of transmembrane segments of receptors such as Aer. These include 5-iodoacetamidofluorescein, 4-acetamido-4-disulfonic acid, disodium salt (AMS), and methoxy polyethylene glycol maleimide, a 5-kDa molecular mass probe that alters the mobility of Aer on SDS-PAGE.
  • Watts, K. J., K. Sommer, S. L Fry, M. S Johnson, B. L Taylor. "Function of the N-Terminal Cap of the PAS Domain in Signaling by the Aerotaxis Receptor Aer." J. Bacteriol. 188. (2006): 2154-2162. ( 3/2006 ) Link...
    Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.
  • Amin, D.N., B. L. Taylor, and M. S. johnson. "Topology and boundaries of the aerotaxis receptor Aer in the membrane of Escherichia coli." J. Bacteriol. 188. (2006): 894-901. ( 2/2006 ) Link...
    Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4'-maleimidylstilbene-2, 2' disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements
  • Watts, K. J., M. S. Johnson, and B. L. Taylor . "Minimal requirements for oxygen sensing by the aerotaxis receptor Aer." Molecular Microbiology 59. (2006): 1317-1326. ( 2/2006 ) Link...
    The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared.
  • Ma Q, Johnson MS, Taylor BL. "Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix." J Bacteriol. 187.1 (2005): 193-201. ( 1/2005 ) Link...
    HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain
  • Watts KJ, Ma Q, Johnson MS, Taylor BL. "Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer." J Bacteriol. 186.21 (2004): 7440-7449. ( 11/2004 ) Link...
    The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 alpha-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.
  • Ma Q, Roy F, Herrmann S, Taylor BL, Johnson MS. "The Aer protein of Escherichia coli forms a homodimer independent of the signaling domain and flavin adenine dinucleotide binding." J Bacteriol. 186.21 (2004): 7456-7459. ( 11/2004 ) Link...
    In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain
  • Herrmann S, Ma Q, Johnson MS, Repik AV, Taylor BL. "PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding." J Bacteriol. 186.20 (2004): 6782-6791. ( 10/2004 ) Link...
    The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fol
  • Herrmann, S., Q. Ma, M. S. Johnson, A. V. Repik, and B. L. Taylor. "PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding." Journal of Bacteriology 186.20 (2004): 6782-6791. ( 1/2004 ) Link...
    The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.