Biomedical subjects

Robert L Kerby

Publications and source records attributed to Robert L Kerby.

13 recordsLinked to original sources

PubMed2006

A C-helix residue, Arg-123, has important roles in both the active and inactive forms of the cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper beta4/beta5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously (Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., Haste, N. M., Korney, A., and Taylor, S. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 45-50). Based on mutagenic evidence, we also propose the basis for the stabilization of the inactive form to be through a salt interaction between Asp-68 and Arg-123.

Arginine↗

PubMed2006

Heme displacement mechanism of CooA activation: mutational and Raman spectroscopic evidence.

The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change.

Bacterial Proteins↗

PubMed2006

Unexpected NO-dependent DNA binding by the CooA homolog from Carboxydothermus hydrogenoformans.

CooA, the CO-sensing heme protein from Rhodospirillum rubrum, regulates the expression of genes that encode a CO-oxidation system, allowing R. rubrum to use CO as a sole energy source. To better understand the gas-sensing regulation mechanism used by R. rubrum CooA and its homologs in other organisms, we characterized spectroscopically and functionally the Fe(II), Fe(II)-NO, and Fe(II)-CO forms of CooA from Carboxydothermus hydrogenoformans. Surprisingly, and unlike R. rubrum CooA, C. hydrogenoformans CooA binds NO to form a six-coordinate Fe(II)-NO heme that is active for DNA binding in vitro and in vivo. In contrast, R. rubrum CooA, which is exquisitely specific for CO, forms a five-coordinate Fe(II)-NO adduct that is inactive for DNA binding. Based on analyses of protein variants and temperature studies, NO-dependent DNA binding by C. hydrogenoformans CooA is proposed to result from a greater apparent stability of the six-coordinate Fe(II)-NO adduct at room temperature. Results from the present study strengthen the proposal that CO specificity in the CooA activation mechanism is based on the requirement for a small, neutral distal ligand, which in turn affects the relative positioning of the ligand-bound heme.

Biochemistry↗

PubMed2005

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.

C-Reactive Protein↗

PubMed2005

CooA, a paradigm for gas sensing regulatory proteins.

The heme-containing transcriptional factor CooA regulates the expression of genes involved in the oxidation of carbon monoxide (CO) in the bacterium Rhodospirillum rubrum. CooA is both a redox sensor and a specific CO sensor, a combination of properties that is unique among heme proteins. Extensive biochemical and genetic analyses, interpreted in the context of a crystal structure of one form of the protein, have allowed the creation of hypotheses concerning the mechanism of CooA activation by CO as well as the basis for its CO specificity. The article details the data in support of these hypotheses and indicates future lines of research.

Bacterial Proteins↗

PubMed2005

Dual roles of an E-helix residue, Glu167, in the transcriptional activator function of CooA.

CooA is a transcriptional activator that mediates CO-dependent expression of the genes responsible for CO oxidation in Rhodospirillum rubrum. In this study, we suggest in vitro and in vivo models explaining an unusual requirement of CooA for millimolar levels of divalent cations for high-affinity DNA binding. Several lines of evidence indicate that an E-helix residue, Glu167, plays a central role in this requirement by inhibiting sequence-specific DNA binding via charge repulsion in the absence of any divalent cation and that divalent cations relieve such repulsion in the process of DNA binding by CooA. Unexpectedly, the Glu167 residue is the optimal residue for in vivo transcriptional activity of CooA. We present a model in which the Glu167 from the downstream subunit of CooA helps the protein to interact with RNA polymerase, probably through an interaction between activating region 3 and sigma subunit. The study was further extended to a homologous protein, cyclic AMP receptor protein (CRP), which revealed similar, but not identical, roles of the residue in this protein as well. The results show a unique mechanism of CooA modulating its DNA binding and transcriptional activation in response to divalent cations among the CRP/FNR (fumarate and nitrate reductase activator protein) superfamily of regulators.

Bacterial Proteins↗

PubMed2004

Changing the ligand specificity of CooA, a highly specific heme-based CO sensor.

The CO-specific heme-based sensor CooA regulates the ability of Rhodospirillum rubrum to grow on CO as an energy source. Only CO triggers the conformational change of CooA essential for the protein to function as a transcriptional activator. A structurally informed mutagenesis, followed by an in vivo screening method, allowed the isolation of a series of novel CooA variants that show very substantial response to imidazole. Compared with wild-type CooA, the ligand selectivity between imidazole and CO had been changed in some variants by roughly three orders of magnitude. Remarkably, different CooA variants also showed the ability to discriminate among imidazole derivatives, strongly implying a mechanism of precise interactions between the affected residues and the various ligands. Although wild-type CooA and imidazole-responsive CooA variants appear to recognize their respective ligands by fundamentally different mechanisms, several lines of evidence suggest that they respond by a similar C-helix repositioning that results in the rearrangement of the DNA-binding domains responsible for specific DNA contact. These results have implications for the molecular basis of both the imidazole responsiveness in the variants and the stringent CO specificity of wild-type CooA.

Carbon Monoxide↗

PubMed2004

Functionally critical elements of CooA-related CO sensors.

CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in Escherichia coli, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from Azotobacter vinelandii and Carboxydothermus hydrogenoformans corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of R. rubrum. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.

Amino Acid Sequence↗

PubMed2004

CO-sensing mechanisms.

Carbon monoxide (CO) has long been known to have dramatic physiological effects on organisms ranging from bacteria to humans, but recently there have a number of suggestions that organisms might have specific sensors for CO. This article reviews the current evidence for a variety of proteins with demonstrated or potential CO-sensing ability. Particular emphasis is placed on the molecular description of CooA, a heme-containing CO sensor from Rhodospirillum rubrum, since its biological role as a CO sensor is clear and we have substantial insight into the basis of its sensing ability.

Amino Acid Sequence↗

PubMed2003

Activation mechanism of the CO sensor CooA. Mutational and resonance Raman spectroscopic studies.

CooA is a CO-dependent heme protein transcription factor of the bacterium Rhodospirillum rubrum. CO binding to its heme causes CooA to bind DNA and activate expression of genes for CO metabolism. To understand the nature of CO activation, several CooA mutational variants have been studied by resonance Raman spectroscopy, in vivo activity measurements, and DNA binding assays. Analysis of the Fe-C and C-O stretching Raman spectroscopy bands permits the conclusion that when CO displaces the Pro2 heme ligand, the protein forms a hydrophobic pocket in which the C-helix residues Gly117, Leu116, and Ile113 are close to the bound CO. The displaced Pro2 terminus is expelled from this pocket, unless the pH is raised above the pKa, in which case the terminus remains in H-bond contact. The pKa for this transition is 8.6, two units below that of aqueous proline, reflecting the hydrophobic nature of the pocket. The proximal Fe-His bond in Fe[II]CooA is as strong as it is in myoglobin but is weakened by CO binding, an effect attributable to loss of an H-bond from the proximal His77 ligand to the adjacent Asn42 side chain. A structural model is proposed for the position of the CO-bound heme in the active form of CooA, which has implications for the mechanism of CO activation.

Amino Acid Substitution↗

PubMed2003

Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains.

Activation of the homodimeric transcriptional regulator CooA depends on the coupling of CO binding at an effector domain heme with the allosteric repositioning of the DNA-binding domain F-helix that promotes specific DNA interaction. By analogy to the homologous cAMP receptor protein (CRP), it has been proposed that effector binding elicits subunit reorientation about their coiled-coil C-helix interface, and that this effector domain reorientation stabilizes the active position of the DNA-binding domains. Here, we describe experiments in which effector-independent "CooA*" variants were selected following randomization of a six-residue portion of the C-helix dimerization domain. Subsequent activity analyses, both in vivo and in vitro, were consistent with a model wherein improved C-helix "leucine zipper" interactions modestly shifted the regulator population equilibrium towards the active conformation, although full activation remained CO-dependent. However, in addition to the improved leucine zipper, maximal CooA* activity required additional C-helix changes which in a WT background decreased normal CO-dependent DNA-binding 100-fold. This seemingly paradoxical combination suggested that maximal CooA* activity depended both on the improved coiled-coil interactions and the decoupling of the signal pathway within the effector domain. Both types of C-helix changes indicate that its repositioning is crucial for the allosteric shift in the inactive/active equilibrium of the DNA-binding domain.

Amino Acid Sequence↗

PubMed2002

The role of the hydrophobic distal heme pocket of CooA in ligand sensing and response.

CooA from Rhodospirillum rubrum is a heme-containing transcriptional activator that becomes activated only upon binding CO. The basis for this specificity has been probed in a CooA variant, termed DeltaP3R4 CooA, lacking two residues adjacent to the Pro(2) heme ligand, which weakens that ligand. DeltaP3R4 CooA can bind imidazole and CN(-), as well as CO, and form a 6-coordinate low spin adduct with each. However, in contrast to the case with CO, imidazole and CN(-) do not stimulate the DNA binding activity of DeltaP3R4 CooA. This result indicates that the CO-specific activation of CooA is not merely the result of creation of a 6-coordinate CooA adduct but that there must be another element to this response. One feature of CooA activation is modest repositioning of the C-helices upon CO binding, so we altered a portion of the C-helix (residues Ile(113) and Leu(116)) located near the heme-bound CO in wild type CooA, and we investigated the effect on CO-specific activation. Surprisingly, the sizes of Ile(113) and/or Leu(116) positions are not critical for CooA activation by CO, disproving a precise interaction between these residues and the CO-bound heme as a basis for the CO activation mechanism and CO ligand specificity. In contrast, hydrophobic residues at these positions contribute to the activation. Some CooA variants altered at these positions in the background of DeltaP3R4 were also found to show low but reproducible activation in response to imidazole binding to the heme. A model for the role of hydrophobicity in CooA activation and specificity is suggested.

Bacterial Proteins↗

PubMed2002

Analysis of the L116K variant of CooA, the heme-containing CO sensor, suggests the presence of an unusual heme ligand resulting in novel activity.

CooA is the CO-sensing transcriptional activator from Rhodospirillum rubrum, in which CO binding to its heme prosthetic group triggers a conformational change of CooA that allows the protein to bind its cognate target DNA sequence. By a powerful in vivo screening method following the simultaneous randomization of the codons for two C-helix residues, 113 and 116, near the distal heme pocket of CooA, we have isolated a series of novel CooA variants. In vivo, these show very high CO-independent activities (comparable with that of wild-type CooA in the presence of CO) and diminished CO-dependent activities. Sequence analysis showed that this group of variants commonly contains lysine at position 116 with a variety of residues at position 113. DNA-binding analysis of a representative purified variant, L116K CooA, revealed that this protein is competent to bind target DNA with K(d) values of 56 nm for Fe(III), 36 nm for Fe(II), and 121 nm for Fe(II)-CO CooA forms. Electron paramagnetic resonance and electronic absorption spectroscopies, combined with additional mutagenic studies, showed that L116K CooA has a new ligand replacing Pro(2) in both Fe(III) and Fe(II) states. The most plausible replacement ligand is the substituted lysine at position 116, so that the ligands of Fe(III) L116K CooA are Cys(75) and Lys(116) and those in the Fe(II) form are His(77) and Lys(116). A possible explanation for CO-independent activity in L116K CooA is that ligation of Lys(116) results in a repositioning of the C-helices at the CooA dimer interface. This result is consistent with that repositioning being an important aspect of the activation of wild-type CooA by CO.

Bacterial Proteins↗