Cook: A heme-containing regulatory protein that serves as a specific sensor of both carbon monoxide and redox state

CooA: A Heme-Containing Regulatory Protein That Serves as a Specific Sensor of Both Carbon Monoxide and Redox State GARY P. ROBERTS, *'1 M A R C V. T H O R S T E I N S S O N , * R O B E R T L. KERBY,* W I L L I A M N . LANZILOTTA, t AND THOMAS POULOS t

*Department of Bacteriology University of Wisconsin-Madison Madison, Wisconsin 53706 t Department of Biochemistry and Molecular Biology Program in Macromolecular Structure University of California-Irvine Irvine, California 92697 I. II. III. IV. V.

VI. VII. VIII. IX. X. XI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CO Oxidation by RhodospiriUum rubrum and Other Microorganisms . . . . . The coo Genes and Their Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Behavior of CooA as a Transcriptional Activator Responding to the Redox State and the Presence of CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Structure of CooA and Its Implications . . . . . . . . . . . . . . . . . . . . . . . . . . A. Comparison of CooA Structure to That of CRP . . . . . . . . . . . . . . . . . . . . B. The H e m e Region of CooA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Model for Activation of CooA by CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CooA as a Redox Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CooA as a CO Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cooperativity of Ligand Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptional Activation by CooA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Recognition Properties of CooA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Direction and Open Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

36 36 38 40 42 42 47 50 52 53 54 56 58 59 60

CooA, the heme-containing carbon monoxide (CO) sensor from the bacterium RhodospiriUum rubrum, is a transcriptional factor that activates expression of certain genes in response to CO. As with other hemc proteins, CooA is 1To whom correspondence should be addressed. Progress in Nucleic Acid Research and Molecular Biology, Vol. 67

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Cop~,Tight O 2001 by Academic Press. All rights of reproduction in any form reserved. 0079-6603/01 $35.00

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GARYE ROBERTSET AL. unable to bind CO when the Fe heme is oxidized, consistent with the fact that some of the regulated gene products are oxygen-labile. Upon reduction, there is an unusual switch of protein ligands to the six-coordinate heme and the reduced heine is able to bind CO. CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts between CooA and RNA polymerase. CooA is therefore a novel redox sensor as well as a specific CO sensor. CooA is a homolog of catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time. The recent solution of the crystal structure of the CO-free (transcriptionally inactive) form of CooA has allowed insights into the mechanism by which both proteins respond to their specific small-molecule effectors. © 2001 Academic Press.

I. Introduction In the past several years, the field of biological sensing of small gaseous molecules such as NO (1-7), 02 (8-14), and H2 (15), in addition to CO (16-18), has shown dramatic progress and involves both prokaryotes and eukaryotes. These sensors must be able to perform some degree of selectivity in sensing their effectors, so that they can trigger the proper biological response and avoid being inhibited or inappropriately triggered by the "wrong" small molecules. Rhodospirillum rubrum can oxidize CO to CO2, and the expression of genes involved in that process are regulated by CooA (CO-oxidation activator). This heine-containing dimer functions both as a redox sensor and as a specific CO sensor. Part of this process involves a highly unusual switch of protein ligands upon reduction of the heme, as well as the utilization of proline as an axial ligand, which has not previously been seen (19). As a member of the CRP/FNR superfamily of regulatory proteins, many of which respond to small-molecule effectors, the solution of its structure in the effector-free form has provided insight into the mechanism by which CooA, and presumably some other members of this family, become activated in response to their effector molecules. The combination of structural information with the results of a variety of mutagenic, spectroscopic, and functional analyses has also revealed important features that underlie its sensing of redox and CO. The analysis of CooA provides an important addition to the understanding of the selectivity in such sensing systems, as well as suggesting the mechanism by which many members of the CRP/FNR superfamily become active for DNA binding.

II. CO Oxidation by Rhodospirillumrubrum and Other Microorganisms CO is found throughout the environment and is the product of chemical decomposition, biological processes, atmospheric reactions, and human

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

37

activity (20). In aquatic environments, the most significant source of CO is thought to be abiotic photooxidation of biological molecules (21, 22), and direct measurements indicate its presence throughout the water column at nanomofar concentrations (21, 23). The removal of CO is through oxidation by radical chemistry in the upper atmosphere or through microbial metabolism, with the latter processes encompassing adventitious mechanisms in which CO is considered a fortuitous substrate, or biochemical systems in which the metabolism of CO (often as an enzyme-bound intermediate) is the connection between oneand two-carbon metabolites of an anabolic or catabolic process. These types of metabolism depend on one of two distinct biochemical mechanisms (24). Aerobic, "carboxydotrophic," organisms exclusively elaborate a molybdenumcontaining hydroxylase that catalyzes the catabolic oxidation of CO and water to CO2 plus reducing equivalents, which ultimately yield H2 through the activity of a hydrogenase. Expression of these systems, such as the 12-gene cox cluster of Oligotr~rpha carboxidovorans, requires the presence of CO, and the cox genes are found adjacent to cbb genes that encode components of the Calvin cycle (25). As yet, the regulatory mechanism of the cox (and homologous) systems has not been described. It will be interesting to compare this CO-dependent regulation, which enables aerobic transcription, with CooA, which controls a low-redox process. Anaerobic CO-oxidizing microbes span a tremendous diversity and encompass methanogenic, acetogenic, and sulfate-reducing organisms. The oxygenlabile enzymatic oxidation of CO to CO2 routinely assayed in vitro is catalyzed by a Ni-containing enzyme that performs, in vivo, the fundamental steps in the interconversion of single-carbon intermediates and acetyl-CoA. While the level of the enzyme may be modestly influenced by the particular carbon substrate upon which the organism is cultivated, its expression does not require the presence of exogenous CO (24). The observation that purple nonsulfur bacteria can anaerobically metabolize CO was first made by Uffen with an organism that is now known as Rubrivivax gelatinosus (26), although R. rubrum is now the best characterized example of the group. Phototrophs express a "hybrid" system: the central Ni-containing enzyme catalyzing CO oxidation is termed carbon monoxide dehydrogenase (abbreviated CODH), and the R. rubrum enzyme is certainly evolutionarily related to those of strict anaerobes, based on predicted primary protein sequences (27). Yet the R. rubrum enzyme, like that of aerobic carboxydotrophs, solely catalyzes CO oxidation to CO,2 and reducing equivalents, which in turn yield He via electron carriers and a specific hydrogenase. The 12-gene coo regulon, aside from that encoding the regulatory protein, is transcriptionally dependent upon the presence of CO. Carboxydothermus hydrogenoformans, a nonphototrophic anaerobe isolated from a volcanic swamp, probably contains a similar CO-oxidation system (28). Perhaps aside from organisms isolated from burning coal piles (29) or volcanic environments (28) where exceptional levels of CO can be expected, the

38

GARYE ROBERTSET AL.

biological niche for most CO-utilizing aerobes and anaerobes is obscure: Measured bulk CO levels, normally in the nanomolar range in aqueous environments and in the range of parts per billion in the atmosphere, are generally well below the Km for CODHs of isolated organisms (30). This is also the case for the R. rubrum coo system. It does not appear to be for "detoxification," both because mutants lacking the coo system are highly tolerant of CO under lab conditions and because R. rubrum is surprisingly capable of coupling the thermodynamically poor anaerobic conversion of CO to CO2 plus H2 to grow at a rate only 20% slower than phototrophic growth in the same medium (31). This complex, specific, and regulated metabolism implies either that R. rubrum naturally experiences periods of exceptional CO exposure or that its native environments are so energy-limiting as to make this system advantageous.

III. The coo Genes and Their Regulation All known coo genes fall into three contiguous transcripts on the R. rubrum chromosome (27, 32, 33), as depicted in Fig. 1. coos encodes the CODH activity and cooF encodes a ferredoxin that forms a tight complex with CooS and promotes its association with the membrane. This membrane association apparently supports efficient electron transfer to the products of the cooMKLXUH operon. Based on sequence similarity and some biochemical characterization (32), the products of the first five genes of this operon form a membrane-associated complex that passes reductant to CooH. In the course of this electron transfer, useful energy is generated, presumably by the generation of a proton gradient, that supports growth on CO as sole energy source (31). CooH is an unusual NiFe hydrogenase in that it is highly tolerant of CO (34), consistent with its role in this CO-oxidizing system, and that it lacks a cleavable C terminus. This C terminus is removed in many Ni-containing hydrogenases upon Ni insertion, but in CooH, the site of normal cleavage is replaced by a stop codon; it is essentially "precleaved" (32). The CooCTJ products are apparently involved in Ni

co /

~

cooM

--

--

I( L X U H

-

C o o A

active~

cooF S C T J

C

inactive

cooA nadC B

F[c. 1. Transcriptional organization of the coo regu]on in R. mbrum. The dark horizontal arrows

indicatethe directionof transcription,nadBC, ORF,and ahpC are not involvedin CO oxidation.

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

39

processing for CooS based on mutant phenotype, similarity to other genes, and substantial biochemical and physiological characterization (33, 35, 36). Both the cooMKLXUH and cooFSCTJ operons appear to be completely under the control of CooA, the CO sensor that is the focus of this review, but cooA has a separate low-level promoter so that it is present in the cell under all growth conditions examined (Y. He and G. P. Roberts, unpublished data). The expression of the coo genes is under the control of CooA, which activates transcription only when it is reduced and binds CO (17, 37, 38). This regulation of coo gene expression can be rationalized in light of our knowledge of the regulated gene products. For example, CO is the only known substrate of CooS, the C O D H encoded by this system (39) [although CooS can also run the reverse reaction (40)], and the regulation of coo expression in the absence of CO appears to be extremely tight in R. rubrum, with C O D H activity, C O D H antigen, and cooFSCTJ mRNA being undetectable in the absence of CO. In the presence of low levels of CO, however, the genes are highly expressed (41), consistent with the potential utilization of this system as sole energy source in the cell. The affinity of CooA (K,I ~ 4 # M ) for CO (M. V. Thorsteinsson et al., unpublished data) is in the same range as the Km of CooS ("~32 #M) (42), so that CooA causes Coos to be synthesized when CO levels allow a reasonable level of enzymatic activity. CooS has also been found to be O2-sensitive, as has CooH, so it is not surprising that the coo genes are also expressed only under anaerobic conditions. Less obviously, Coos is maximally active at a redox poise of - 3 0 0 mV and below (J. Heo and P. W. Ludden, personal communication), and the behavior of CooA is consistent with this fact. CooA is competent to bind CO only when its heme is reduced, and the reduction and oxidation midpoint potentials have been reported to be - 3 2 0 and - 2 6 0 mV, respectively (43), reflecting the different initial CooA species present in each redox titration. Although this comparison is certainly simplistic and we have not determined the internal redox state of R. rubrum cells expressing the coo genes, it serves to suggest that regulation of CooA activity might be sufficient to explain the observation that reducing culture conditions are necessary for consistent high-level coo expression in R. rubrum (31). In the absence of CO or in the presence of oxidizing conditions, only cooA is expressed. However, under reducing conditions in the presence of CO, cooFSCTJ and cooMKLXUH become very highly expressed, with the former being expressed at a level approximately fivefold higher than the latter. Oddly, in the presence of CO, some portion of the cooFSCTJ transcript appears to read through into cooA, and Western analysis has shown an increase in CooA accumulation in R. rubrum in the presence of CO (Y. He and G. P. Roberts, unpublished data). The physiological reason for this readthrough, if any, remains unknown. Given the ability of the coo system to provide the sole energy source for the cell, one might expect its expression to be regulated by the energy status of the

40

GARYE ROBERTSET AL.

cell. More specifically, one might predict that the cooFSCTJ operon would be expressed under anaerobic conditions in the presence of CO, since the expression of these genes should support "detoxification" of CO, but that the cooMKLXUH operon would have have an additional layer of regulation that reflects energy needs. However, as we have shown that virtually all of the reductant generated by CooS flows through CooH, the hydrogenase, it appears possible that the proteins encoded by the latter operon are necessary for the efficient consumption of the reductant (31). Also, as both operons are expressed in the presence of light, an excellent energy source, there seems to be no reason to propose additional regulatory inputs, nor is there any molecular evidence for the involvement of other regulatory factors. In general, the highest expression, as measured by protein accumulation, appears to be that ofcooFS, suggesting that some attenuation reduces the transcription into cooCTJ (Y. He and G. P. Roberts, unpublished data). Relatively little has been done to study the mechanism by which the coo genes are turned off upon the exhaustion of CO. The addition of 02 to a cooexpressing culture leads to the fairly rapid cessation of coo expression (41), as would be expected, but unanswered questions remain about the precise response to CO exhaustion. Expression would presumably cease only when no CO-bound CooA remained in the cell; however, this exhaustion might take a very long time, as CooA is a fairly abundant regulatory protein (Y. He and G. P. Roberts, unpublished data) and the kinetic rate of dissociation for CO bound to heme proteins (e.g., sperm whale myoglobin) is typically extremely long (with a rate of dissociation of "~1 rain) (44). This "problem" would be exacerbated by the increase in CooA levels in the presence of CO due to transcriptional readthrough. The resolution of this paradox will require further physiological analysis of R. rubrum under these changing conditions.

IV. General Behavior of CooA as a Transcriptional Activator Responding to the Redox State and the Presence of CO CooA is a homolog of both CRE a cAMP sensor in E. coli that regulates genes whose products are involved in carbon utilization (reviewed in Refs. 45 and 46), and FNR, an E. coli protein involved in regulation of gene expression in response to anaerobiosis (11, 13, 47, 48). Numerous other homologs have been identified through sequence comparison, with biological roles such as control of virulence (49, 50), although CRP and FNR are by far the best studied. Interestingly, although both FNR and CooA are sensors of the redox state of the cell, they do so by completely different mechanisms, and CooA actually appears to be more similar to CRP in general behavior. Both CooA and CRP

CooA:A CO-SENSING TRANSCRIPTIONALFACTOR

41

are homodimers under all conditions (17, 51, 52) and undergo a conformational change upon effector binding. With C RP, binding of the effector cAMP induces a conformational change within the dimer, which is thought to cause the DNAbinding domains to reorient themselves so that they can simultaneously interact with one-half of a palindromic DNA target sequence (see Ref. 53 for a recent method and a summary of previous approaches). In contrast, FNR appears to be primarily regulated through a monomer-dimer transition, the formation of the DNA-binding dimerie form being caused by the formation of Fe4S4 centers only under anaerobic conditions (47, 48). Dimeric CooA contains a protoheme moiety in each monomer that is the site of CO binding (17, 38, 54, 55). It can exist in three states: (1) When the heine is oxidized, CooA is incapable of binding CO or DNA; (2) when the heine is reduced, CooA cannot bind DNA, but can bind CO; and (3) in the reduced, CO-bound form, CooA is competent to bind DNA. The details of these transitions, as well as the structural features that affect this behavior, are described in detail below. Upon reduction, a required step for CO binding, CooA undergoes a conformational change, as detected on native gels (54), which in part reflects a highly unusual ligand switch (38, 54) by which one of the protein ligands to the heine (Cys-75) is replaced by another (His-77). As detailed in Sections V,B and VI, this ligand switch involves a rearrangement of the heme with respect to the protein. It is likely that a role of this switch is to set the proper redox poise of the protein, although this issue has not been well-studied. Upon CO binding to reduced CooA, another conformational change is apparent on native gels (54), and it is this change that leads to a form of CooA that is competent to bind DNA and to promote transcriptional activation in vitro (17, 54). Further information on the DNA sequences bound and the interaction of CooA with RNA polymerase is provided in Sections IX and X. The crystal structure of the reduced, effector-free (reduced but lacking CO) form is described in the following section, as are hypotheses concerning the conformational change upon CO binding. Briefly, the binding of CO displaces the Pro-2 ligand to the heme, thereby triggering the conformational activation. CO, but no other small molecule, leads to the activation of CooA so that it is a specific CO sensor. This is particularly significant, as another well-studied heinecontaining sensor, FixL, responds to a variety of small molecules in vitro (9, 56, 57). Similarly, soluble guanylyl cyclase might be able to respond to CO under some conditions (16, 58), in addition to its well-studied response to NO (1-5). In the case of CooA, however, 02 oxidizes the heme, whereas cyanide, azide, and imidazole fail to bind at all to either oxidation state of the protein (,54). While NO is capable of binding the heine, it displaces both protein ligands and CooA fails to become active (59), suggesting that activation requires the displacement of the appropriate protein ligand together with the retention of the other.

42

GARY P. ROBERTS ET AL.

The cooperativity of CO binding to CooA (see Section VIII) is likely to be of physiological significance, and it is interesting to consider effector binding in CRP. CRP appears to bind its effector, cAMP, with negative cooperativity (60, 61 ), although claims of positive cooperativity have also been made (62, 63). At physiologically reasonable levels of cAMP, one would expect CRP to accumulate with a single cAMP bound; it is therefore unsurprising that this form of CRP appears to have the highest affinity for its DNA target site (60). Rather recently, the structure of CRP was determined with four cAMP molecules bound per CRP dimer (64), although the physiological significance of this form remains unclear and will not be considered further here. However, neither the role of this negative cooperativity for cAMP/CRP in vivo nor its mechanism is understood. In the case of CooA, the functionality of a dimer with a single CO bound is unknown.

V. The S~ructureof CooA and Its Implications The structure of the effector-free form (reduced but without CO) of CooA has been solved at 2.6 A resolution (19) and reveals several striking features. First, each monomer of CooA contains a heme with a novel ligand, which is Pro-2 at the N terminus of the other protein monomer. Proline has not previously been identified as an axial ligand to a heme, and its properties as a ligand are therefore of substantial interest. Second, the structure of the reduced CooA dimer is highly asymmetric (Ref. 19 and Fig. 2A; see color insert) with the two different monomer forms designated A and B. It is clear that this asymmetry is a property of the packing in the crystal lattice. Form B makes no contacts with other dimers in the crystal lattice, while form A is confined to the observed conformation by contacts from three other molecules in the crystal lattice (19). It is therefore our working hypothesis that effector-free CooA in solution is likely a fully symmetric dimer of form B. Although it is clear that there are at least three physiologically significant structures of CooA (oxidized, reduced, and reduced + CO), only the structure of the reduced, effector-free form has been solved. Nevertheless, a number of important insights into the total behavior of CooA can be drawn, in part because of the existence of the structure of the effector-bound form of CRP, both in the presence (65, 66) and absence (67) of DNA. The following sections draw comparisons between the structures of effector-free CooA and effector-bound CRP, then focus on the residues in the immediate vicinity of the heme, and finally propose a mechanism of activation of CooA by CO.

A. Comparison of CooA Structure to That of CRP Because the CooA structure represents its effector-free form, it is useful to compare this CooA structure to the various structures of CRP, all of which

,-,

D

FIG. 2. (A) Structure of the reduced form of CooA (19). The monomer shown in turquoise is referred to in the text as form A, while the monomer in brown is form B. The red molecule is the heme on each monomer. (B) Structure of cAMP-bound CRP (65). The red molecule is cAME (C) C Helix movement upon activation. The panel shows the comparison of the C helix of effectorbound CRP (in orange) compared to that of effector-free CooA (in blue). The relative positions of the heme (in blue) and the cAMP (in orange) are also shown. (D) The vicinity of the heine of the reduced form of CooA. Residues 74-78 are displayed, with His-77 centered over the heme and serving as the ligand. The position of Cys-75 is indicated to the left of His-77.

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

43

are in the effector-bound form (including CRP bound to DNA). Figure 2B (see color insert) shows the effector-bound form of CRP, which shows only a modest rotational asymmetry between the two subunits (67), although DNA binding renders the dimer highly symmetric (65, 66). The site of cAMP binding is in the center of the effector-binding domain, immediately adjacent to the long ot helices, termed the C helices (Fig. 3), that form the dimer interface. Indeed, there is an interdigitation of residues in this portion of CRP, such that a residue (Set-128) in one monomer makes contact with the cAMP bound to the other monomer (67). The conformational change in CRP upon cAMP binding is unknown, although a variety of approaches indicate that the change is significant (53, 68, reviewed in Ref. 45). In the absence of cAMP, CRP has much poorer affinity for its specific DNA target (60). Although the structure of effector-bound CooA has not been solved, that structure is likely to be rather similar to that of effector-bound CRP, as both proteins bind structurally similar DNA sites and have at least some similarities in their interactions with RNA potymerase (Section IX). There are several fairly

FIG. 3. Left: Monomerof form B of CooA;right: monomerof CRE For each structure, the letters are adjacentto the appropriateu helices,whilethe numbersare adjacentto the appropriate fl sheetsof the two monomers.

44

GARYE ROBERTSET AL.

striking differences between effector-bound CRP and form B of effector-free CooA: (1) The C helix of CooA is extended by means of a fusion of the C and D helices, relative to that of CRP. (2) The relative positions of the C helices within the effector-binding domain in CooA is distinct from that of CRP. (3) There are a number of different contacts made by residues in the "hinge" region, which is between the DNA-binding and effector-binding domains of the two proteins. (4) There is a completely different orientation of the F helices, which create the sequence-specific contacts with DNA, in the two proteins. (5) The positions of the effector-binding sites are similar, but not identical, in CRP and CooA. Each of these issues is addressed in turn below. 1. EXTENSION/FUSIONOF THE C HELIX OF EFFECTOR-FREE CooA Form B of CooA achieves its extended configuration, relative to that of CRP, because of the extension of the long C helix that serves as the dimer interface in both CooA and CRP (Fig. 2A,B). That extension is actually due to the orientation of helix D of the DNA-binding domain (Fig. 3) relative to its position in CRP, such that the D helix fuses with the C helix. In effectorbound CRP, the D helix lies close to the upper surface of the effector-binding domain in an orientation relative to that of the C helix that is more than 140 ° different than in form B of CooA. Effector-bound CRP thus provides a number of contacts between the DNA-binding and effector-binding domains, with both the D and E helices of the DNA-binding domain providing contact with/3 sheet 5 (Fig. 3) of the effector-binding region. In contrast, there is relatively little surface contact between these two domains in form B of CooA, the only apparent interaction being between the loop between the E and F helices of the DNA-binding domain and the tip of the loop formed by/3 sheets 4 and 5 (the "4/5 loop"), an element of the effector-binding domain. Although it is not clear that effeetor-free CRP has a similar extended structure, that hypothesis is consistent with the data, as neutron scattering experiments (68) have indicated that effector-free CRP is significantly extended relative to effector-bound CRP. If both the effector-bound and the effector-free forms of CRP and CooA were shown to be similar, it would imply that the mechanism of activation of the two proteins was also likely to be similar. It is tempting to hypothesize an additional sort of stabilizing interaction that might also be present in a dimer of form B CooA namely, interaction between the two DNA-binding domains themselves. Such an interaction is difficult to assess at present because the structure is defined only through Ala-213, so that the position of the C-terminal 9 residues is unknown (19). However, the structure suggests that these residues in each monomer might be oriented so that they could interact. In any event, it is clear that there are substantial differences in the positions of interaction among the various domains within the dimers of

CooA:ACO-SENSINGTRANSCRIPTIONALFACTOR

45

effector-bound CRP and effector-free CooA that reflect differences in activation state rather than primary protein sequence. 2. THE RELATIVEPOSITIONS OF THE C HELICESWITHINTHE EFFECTOR-BINDING DOMAINCHANGESUPONACTIVATION Besides the dramatic conformational differences noted above, there is a more subtle but very significant difference in the relative position of the two C helices with respect to each other in the two proteins (19). While each C helix of CooA can be aligned with its homolog helix in CRP, the relative positions of the two C helices in the two proteins is quite different (Fig. 2C; see color insert). There is a bending and rotation of the C helices with respect to each other such that (in effector-bound CRP as compared to effector-free CooA) they move apart at the bottom of the C helix with no relative movement at the fulcrum, represented by Leu-130 of CooA (Leu-134 of CRP). This has the effect of repositioning the two C helices such that they have different regions of closest contact in the two proteins. Indeed, along the entire length of the C helices in both CooA and CRP, there is a suboptimal leucine zipper and the repositioning upon activation has the effect of changing the regions where these two leucine zippers interact. The general issue is discussed further in Section V,C,1. 3. DIFFERENTIALCONTACTSMADE BYRESIDUES IN THE HINGE REGION OF CRP AND CooA The "hinge" region of CRP (Fig. 3), centered on Asp-138, is the boundary between the DNA-binding and the effector-binding domains of that protein (67). The fusion of the C and D helices (relative to their position in CRP) in form B of CooA directly affects the CooA region analogous to the hinge region of CRP, and it is not surprising that residues in the vicinity of that reorientation make rather different contacts in the two proteins. More interestingly, the specifics of these contacts appear to be important in stabilizing each of the respective conformations (19). Two residues are particularly relevant: Phe-132 of CooA (homologous to Phe-136 of CRP) and Arg-138 of CooA (homologous to Arg-142 in CRP). In CooA, Phe-132 interacts with the F helix of the DNAbinding domain, while Phe-136 of CRP makes contact with the 4/5 loop of the effector-binding domain. The region of interaction in the 4/5 loop is retained in CooA, suggesting that such an interaction might also be relevant to effectorbound CooA. The other residue, Arg-138, interacts with the 4/5 loop in CooA (of the other monomer), but its homolog in CRP, Arg-142, interacts with the DNA backbone. The actual functional importance of these interactions remains to be examined, but the very different role of these residues in the effectorbound and -free forms implies that their reorientation upon activation is central to the activation mechanism.

46

GARYP. ROBERTSET AL.

4. THE POSITION OF THE F HELICES THAT CONTACT DNA ARE DRAMATICALLYDIFFERENT IN EFFECTOR-FREE CooA AND EFFECTOR-BOUND CRP Another implication of the CooA structure (irrespective of which form of CooA is analyzed) is the fact that the F helices, which are necessary for direct interaction with the specific DNA target sequence in CRP and must certainly perform a similar role in CooA, are both oriented approximately 180 ° away from their position in CRP. That is, in effector-free CooA, they "face" the effector domain, which would make DNA interaction virtually impossible. Given that both CRP and CooA show a very low affinity for DNA in the absence of effector (17, 45), one might assume that this low affinity reflects a small fraction of the proteins that have reoriented their DNA-binding domains spontaneously and transiently without effector. It is a formal possibility, however, that low affinity for nonspecific DNA reflects interactions with other regions of the proteins. In any event, the positioning of the DNA-binding helices in effector-free CooA would seem to be of biological importance and will need to be further examined. 5. THE POSITIONS OF THE EFFECTOR-BINDING SITES ARE SIMILAR, BUT NOT IDENTICAL, IN CRP AND CooA

The heroes of CooA lie somewhat below the analogous position in CRP where cAMP binds (Fig. 2A, B), although we believe that it is important that both the heme and cAMP interact directly with portions of the C helix. A hypothesis for the role of effector-mediated perturbation of the C helices in activation is discussed in Section V,C. The heme occupies a pocket that does not exist in CRP, but seems to be accommodated by the deletion of eight residues in CooA relative to CRP (approximately residues 73-80 of CRP) (19). It also appears that there is a small rotation, as viewed down the axis of the C helix, of part of the effector domain away from the C helix to make space for the heine. From a protein evolutionary standpoint, it would be interesting to know if CooA evolved from a protein that lacked heine and therefore what structural changes were essential to create a pocket for heine binding. In addition to the structural differences between CooA and CRP noted above, a number of other differences can be detected between the two forms of these proteins (19) that are reflective of the substantial conformational differences. Some of these differences no doubt reflect critical differences between effector-bound and effector-free CRP/CooA, while others merely reflect differences between CooA and CRP themselves. Further experimentation will be necessary to distinguish between these two possibilities for any observed difference in the two structures in hand. The structural differences also fail to identify the mechanism by which binding ofeffector triggers the change between the two conformations, and the hypotheses for this trigger are described in Section V,C.

CooA: A CO-SENSING TRANSCRIPTIONAL FACTOR

47

B. The Heme Region of CooA The structure of reduced, effector-free CooA confirmed the role of His-77 as one ligand to the ferrous heme iron, as suggested by previous mutagenic and spectroscopic studies (38, 54). Another aspect of the structure of this side of the heme was surprising, however, in that Cys-75, which certainly replaces His-77 as the ligand in the oxidized form (69), is approximately 4.8 ~_ from the heme Fe (Fig. 2D; see color insert). This implies that there must be a conformational change in the protein, a movement of the heme, or both, in the course of the reversible oxidation and reduction of the berne. Under the assumption that there is at least some heine movement, the present structure predicts that the heme would "move into" the effector domain upon oxidation. Because the heine remains low-spin, six-coordinate under both conditions, this implies that the Pro-2 ligand on the other side of the heme is either extremely flexible in its position or that a ligand switch takes places on that side as well upon oxidation/reduction. Currently, we favor the former hypothesis because of the results described in Section V,B,3. His-77 appears to be relatively free of contacts with other residues in the reduced form, with the exception of Cys-75 and Asn-42; the role of this latter residue in activation of CooA is unknown, but its location adjacent to His-77 suggests that it is likely to be of some importance. The axial ligand on the other side of the heme in reduced CooA was one of the major surprises revealed by the structure (19). That ligand is Pro-2, the N-terminal residue from the other monomer of the dimer. Proline has not been previously identified as a heine ligand, because the secondary amine would be unavailable to serve as a ligand when involved in a typical peptide bond. However, as the N-terminal residue (following posttranslational removal of Met-1), the N ofproline appears to serve as a reasonably strong-field ligand, consistent with the observation that known strong-field small-molecule ligands such as cyanide are unable to displace it. The spectroscopies of MCD, RR, and EPR were not able to distinguish the Pro ligand from the very common His ligation, suggesting that Pro appears as a "neutral nitrogen donor" in such spectroscopic analysis (55, 70, 71 ). As described below, while Pro is a highly unusual ligand, it does not appear essential for CO responsiveness of CooA, and the precise role of this region of CooA for a response to CO needs to be determined (72). The exchange of N-terminal arms between subunits of the CooA dimer is an example of"domain swapping" whereby subunits of an oligomeric protein are linked together by the exchanged domains. The presence of a proline at the position where the intercalating domain extends from its monomer, presumably Pro-14 in CooA, is often crucial for proper configuration of the exchanged arm (73). It is probably of functional importance that the N terminus of CooA, ending with Pro-2, appears to be relatively unconstrained in the structure. This lack of constraint might provide the flexibility necessary to allow the

48

GARYE ROBERTSET AL.

movement of the heme that almost certainly takes place during oxidation reduction (19, 38, 54). The importance of these ligands for CooA function has been explored by a variety of mutagenic approaches and analyses, which will be briefly summarized. 1. CYS-75 Is NOT IMPORTANTFOR THE RESPONSE TO CO Biochemical and spectroscopic analyses of variants altered at position 75 have shown that Cys-75 (1) is important for heme stability in oxidized CooA (54); (2) is not critical for a functional response to CO, as C75S and C75A CooA have significant activity in vivo (38, 54); (3) is presumably important for setting the proper redox poise of CooA; and (4) can also be partially replaced by an unknown adventitious ligand in the oxidized state of variants that lack Cys-75 (54). Interestingly, Cys, Ser, and Ala appear to be the only acceptable residues at position 75 that yield functional CooA, as these were the only substitutions found when the codon was randomized and the resultant library was screened for a response to CO in vivo (M. Conrad and G. P. Roberts, unpublished data). The similar size of these residues suggests that this might be the critical requirement, and preliminary results suggest that larger residues at this position might affect protein stability, possibly through effects on heine stability. Besides serving as an axial ligand for oxidized CooA, the residue at position 75 therefore appears to have some additional structural constraints. 2. HIS-77 Is CRITICAL FOR ACTIVATION OF CooA IN RESPONSE TO CO CooA variants altered at position 77 (1) accumulate berne-containing CooA that is reasonably stable (38, 54); (2) bind CO but are unable to activate transcription in vivo and are unable to bind DNA in vitro (using an assay of fluorescence polarization with tagged target DNA; M. V. Thorsteinsson et al., unpublished data); (3) are perturbed in their UV/Vis spectra in the reduced but not the oxidized form (38, 54); (4) are able to bind cyanide in the reduced form (in contrast to all forms of wild-type CooA, which fail to bind cyanide) (74); (5) typically bind cyanide with positive cooperativity, although the nature of the cooperativity depends on the residue at position 77, as well as on the small molecule ligand (Ref. 74 and M. V. Thorsteinsson et al., unpublished data); (6) are missing a spectroscopic species detected in the oxidized form of wild-type CooA (when Cys-75 is the ligand), consistent with an interaction between His-77 and Cys-75 in wild-type CooA (54); (7) are perturbed in their redox-mediated ligand switch, where they appear to be significantly more difficult to reduce (54, 70); and (8) accumulate to varying degrees as six-coordinate, low-spin species in the reduced form, which implies the presence of at least one adventitious ligand that can replace His-77 (54, 70, 74).

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

49

These various results indicate that His-77 is important for the proper poise of the redox-mediated ligand switch and absolutely critical for the conformational change that alows DNA binding in response to CO binding. As it has very recently been shown that Pro-2 is displaced by CO binding in CooA (73a) the importance of His-77 is presumably to properly position the heme for which the trans ligand has been released by CO. Note that variants lacking His at position 77 are sixcoordinate, low-spin to varying degrees when reduced (70, 71), so there must be an adventitious ligand on that side of the heme that can replace the missing His-77. Although this adventious ligand is apparently strong enough to create a low-spin heme, it must position the heine incorrectly upon CO binding, and this mis-positioning presumably is critical for activation, but of modest influence on eooperativity. The identity of the adventitious ligand is presently unknown. 3. PRO-2 IS OPTIMAL, BUT NOT CRITICAL, FOR HEME ACCUMULATION AND THE RESPONSE TO CO Given the uniqueness of Pro-2 as a heme ligand, we expected that this residue would be central to CooA function. However, variants altered in the Pro-2 region (including those with longer and shorter N termini, as well as with different terminal residues) often accumulate reasonable amounts ofheme-containing CooA that responds to CO both in vivo and in vitro (72). These variants appear generally normal in UV/Vis spectra for both the reduced and reduced + CO forms, suggesting that some adventitious ligand is replacing Pro-2 in at least the former case, when it is known to serve as a heine ligand. However, these variants are significantly altered in the oxidized form, where they have a mix of five- and six-coordinate species. When one example, P2Y, was purified and characterized, it displayed an affinity for CO that was slightly greater than that of wild-type CooA and a modest decrease (10-fold) in affinity for DNA in vitro in the presence of CO (72). These results support the following conclusions about Pro-2 in CooA. (1) There is apparently an adventitious ligand that can replace Pro-2 in the reduced form of CooA. The structure does not suggest an obvious candidate, other than the N terminus created by the replacement of Pro-2. This possibility is attractive, since it should have properties generally similar to Pro and would be present in all variants. However, a deletion of residues Pro-3 and Arg-4 has approximately the same properties as other Pro-2 region variants (R. L. Kerby et al., unpublished data) and it is unclear if the N terminal region is sufficiently flexible to reach the heine iron when shortened by two residues. (2) Pro-2 is likely to be the ligand in the oxidized form of CooA, as the spectroscopy of this form is severely perturbed in Pro-2 variants. (3) CooA can function with a wide variety of residues at the N terminus, consistent with the great flexibility of the protein in the vicinity of the heme and the relative unimportance of Pro-2 in the CO-activated state.

50

GARYEROBERTSETAL.

Surprisingly, given the apparent lack of a critical importance of Pro-2 to activation, some other residues near the N terminus seem to be more critical for the accumulation of CO responsive CooA in vivo (72). Arg-4 is of some importance for accumulation of heme-containing CooA, and because its amino group is positioned very close to one of the proprionate groups of the heme, it is possible that this interaction is important for heme stability (19). Phe-5, which fills a hydrophobic pocket near the bottom of the effector-binding domain, is also necessary for CooA accumulation, and this is probably due to effects on protein stability. Finally, Ash-6 lies at the end of an a helix and forms a hydrogen bond with Asn-9, which might be the basis for its importance. The effects of the alteration of Pro-2 on activation of CooA by CO are particularly surprising in light of the demonstration that it is the ligand displaced by CO (73a). This suggests that either the displaced Pro-2 has relatively little role in activation or else some other residue can serve that role satisfactorily. The obvious model is therefore that the precise positioning of the heme by His-77 ligation is critical for activation. We therefore favor a hypothesis that the removal of the Pro-2 "tether" frees the heme to interact with the C helices and affects the relative positioning of these helices, and that this serves as at least one portion of the activation mechanism.

C. Model for Activation of CooA by CO The primary and secondary structures of CooA and CRP are similar enough to provide a basis for hypotheses about the nature of activation of both proteins by their effectors. It is highly likely, for example, that CooA assumes a structure upon CO binding that is rather similar to that of cAMP-bound CRP, as CooA must interact both with a similar DNA sequence and with several similar regions of RNA polymerase to stimulate transcription activation (see Section IX). For both proteins, it is easiest to consider only two states, active and inactive, in an equilibrium. Without effector, this equilibrium is substantially toward the inactive form, as evidenced by the very low, but detectable, activity of both proteins in the absence of effector (17, 45). The binding of effector shifts that equilibrium either by destabilizing the inactive form, stabilizing the active form, or both. An important fact that must be addressed in any hypothesis of activation is that the DNA-binding domains in both proteins lie far from the site of effector binding so that there cannot be a direct interaction between the effector-binding site and the DNA-binding domain. The comparison of the structures of CRP and CooA suggests that the C helix positioning is central to the response to effector for both proteins. Of the differences between the CRP and CooA structures noted in Section V,A, the difference in positioning of the C helices provides the most obvious direct link between the sites of effector binding and the rest of the proteins. The general hypothesis is therefore that effector binding causes a shift in the relative position

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

51

of the C helices, which changes in regions of closest proximity along the leucine zipper that forms the dimer interface. This change in C helix position would potentiate the changes (noted in Section V,A) in the hinge region, eliminating some of the stabilizing factors in the inactive form of the proteins and allowing new interactions that stabilize the active form. This is actually a slightly more specific version of the model of activation proposed previously for CRP in which cAMP binding was proposed to change the "alignment" of the effector-binding domains, although the nature of the effector-free form of CRP could not be guessed from the available data (45). This hypothesis for activation appears to be consistent with the available data on CRP, particularly the data on effector-independent variants (so-called CRP* variants) and those variants altered for cooperative binding of effector. Mutations leading to the CRP* phenotype fall in two general regions (45, 46): in the vicinity of the hinge itself and in the vicinity of the cAMP binding site. Those in the former category presumably "bypass" the normal activation process by either stabilizing the active form or destabilizing the inactive form of the protein, which is consistent with the proposed model. The class of CRP* mutants affecting the cAMP binding site is exemplified by changes in Thr-127 and Ser-128, which affect both the cooperativity of cAMP binding and effector-independent activity (63, 75, 76). It is striking that many of these variants do not significantly affect cAMP affinity, so that the position of these residues at the cAMP-binding site might be coincidental to the phenotypes observed. Instead, it is likely that these changes affect the quality of the leucine zipper in this region. The T127L substitution in CRP is particularly effective at altering the phenotype and also improves the predicted leucine zipper motif. In the case of CooA, we have also found that altering the leucine zipper in the 121-126 region of CooA (homologous to the 125-130 region of CRP) toward that ofa"stronger" leucine zipper has the effect of creating effector-independent CooA variants (termed CooA* by analogy), consistent with the above hypothesis (R. L. Kerby, unpublished data). Indeed, one of the strongest CooA* variants we have obtained is a double mutant, C123L/M 124T, which affects the homologs of CRP residues 127 and 128. This variant has the property of being active under all conditions (oxidized, reduced, and reduced plus CO), although the precise level of activity does show some dependence on the condition examined. The number of well-characterized CooA* variants is low and does not provide a critical test of the hypothesis. Unfortunately, the putative CooA* variant M131L, which has been published several times (38, 77, 78), may not be informative. This variant, which affects the hinge region, has been proposed to be approximately 18 times as active as wild-type CooA in the presence of CO, which would be extraordinary given the apparently efficient activation of wild-type CooA by CO. However, we have created the identical substitution in our lab, and it has the more reasonable phenotype of only a modest CO activation (R. L. Kerby and G. P. Roberts,

52

GARYP. ROBERTSET AL.

unpublished data), consistent with other variants at that position that have also been published (77). We assume that the causative mutation for the high levels of activity is not the M 131L substitution, but rather a mutation in the lacZ reporter gene or elsewhere in the reporter strain. In general then, what data exist for CooA are consistent with the proposed activation mechanism. It is also interesting that FNR has some phenotypically interesting mutations in the C helix region as well. FNR apparently undergoes a monomer--dimer transition in the course of activation, reflecting the synthesis of Fe4S4 clusters in the effector-binding domain (47, 48); thus, its activation mechanism might be expected to be fundamentally unlike that of CRP and CooA. Nevertheless, a D154A substitution (where D154 is the homolog of T127 of CRP and C123 of CooA) or a D154V substitution creates effector-independent variants, in this case independent of the need for reducing conditions and the resultant Fe4S4 clusters (13, 48, 79). While an Ala would not be expected to create a strong leucine zipper, the Asp found in wild-type FNR should be highly deleterious for such a zipper. How might CO binding to the heine of CooA cause this perturbation of the C helices? Our best current hypothesis is that CO binding allows a repositioning of the heine and this repositioning results in the movement of the bottoms of the C helix away from each other, which in turn potentiates the various conformational changes noted above. The specifics of the role of heme positioning in C helix movement is a major issue in understanding the behavior of this protein.

VI. CooA as a Redox Sensor CooA will not bind CO until the heme is reduced, and the midpoint potential of that reduction has been reported to be -320 to -260 mV for reduction and oxidation, respectively (43). As described in Section I1, this potential makes physiological sense in light of the properties of CooS, the CODH of the system. CooA sets the proper redox poise in part through an unusual ligand switch, whereby Cys-75 is replaced as a ligand by His-77. While Cys-75 as thiolate is presumably a better ligand to the oxidized heme than is the imidazole of His-77, and His-77 should be a better heme ligand in the reduced state than is the thiolate of Cys-75, the details of this ligand-switch mechanism are poorly understood. Irrespective of these details, however, the mechanism certainly must be different from the O~/redox sensors that have been found, such as FNR (13, 47, 48, 80), FixL (10, 56, 57), OxyR (81), and DOS [a putative 02 sensor from E. coli, (•4)], as these either do not involve a heme or, in the case of FixL and DOS, actually sense 02 by directly binding it. In this article we have referred to "redox-sensing" somewhat loosely, implying rather more than we actually know. Specifically, the "signal molecule"

CooA:ACO-SENSINGTRANSCRIPTIONALFACTOR

53

that is actually sensed by CooA in the cell is unknown; thus while the regulation "reflects" the redox state of the cell, it is highly likely that a specific small molecule is actually sensed. There certainly are small molecule redox couples, such as NAD/NADH, with midpoint potentials that might be appropriate for CooA. We cannot rule out some sort of heine reductase intermediary, although the fact that no such factor is encoded by the coo regulon of R. rT~brum, as well as the functionality of the coo system when moved to other organisms such as R. sphaeroides and E. coli (82), suggests that this is less likely. In vitro, CooA is readily reduced by small molecules such as methyl viologen, sodium dithionite, and titanium citrate, and is oxidized by potassium ferricyanide; however, physiologically significant small molecules need to be assessed for their ability to reduce CooA in vitro. Although the precise mechanism of redox response of wild-type CooA is unclear, some very interesting variants with different responses to oxidizing conditions have been found. Perhaps the most striking is M124R (M124K being generally rather similar). Cells containing M124R CooA behave like cells with wild-type CooA under anaerobic conditions (no CooA activity) and anaerobic conditions with CO (high CooA activity) (R. L. Kerby, M. V. Thorsteinsson, and G. P. Roberts, unpublished data). However, when grown aerobically (and without CO), cells with M124R CooA display significant levels of CooA activity, in striking contrast to cells with wild-type CooA. Consistent with these observations, purified reduced M124R CooA shows normal UV/Vis spectra in the presence and absence of CO and normal DNA binding only in the presence of CO. When oxidized, however, purified M124R CooA binds DNA in vitro in the absence of CO, and UV/Vis and EPR analyses indicate that there is a mix of five- and six-coordinate species in the oxidized state. Because this proportion changes toward the six-coordinate form at elevated pH, it has been possible to show that the five-coordinate form is the source of the activity. There are no obvious hints from the crystal structure why this substitution would have these properties; but irrespective of mechanism, M124R CooA is, in a sense, a sensor of"oxidizing conditions." The fact that CooA senses redox by a mechanism unlike that of other studied sensors, coupled with the availability of CooA variants altered in redox sensing, makes this a highly interesting aspect of CooA function that merits substantially more attention in the future. This variant is suggestive of the fascinating range of behaviors that CooA variants will be capable of and whose eventual analysis will address larger issues of heine protein function.

VII. CooA as a CO Sensor As noted previously, wild-type CooA responds exclusively to CO, as other small molecules (such as cyanide, azide, and imidazole) either fail to bind to the

54

GARYE ROBERTSET AL.

heme (54) or, in the case of NO, displace both heine ligands and fail to cause an active conformation of CooA (59). The failure of NO, which displaces both protein ligands to the berne, to activate CooA is consistent with the hypothesis that the precise positioning of the heine is essential to the activation process, and certainly the protein ligand that remains bound trans to the CO would be expected to be important for this positioning. The failure of CooA to bind other small molecules might reflect any of several factors: (1) The small molecules are not of sufficient field strength to replace the protein ligands; (2) the small molecules might be stericallyprecluded from reaching or binding to the heme because of a restricted heme pocket; or (3) charge repulsion might restrict the ability of charged small molecules to bind. The first possibility appears to play a role, as CO and NO, the only two small molecules that bind to wild-type CooA, have greater field strength than any of the other small molecules. However, the heme appears to be generally exposed, suggesting that steric hindrance is not likely to be a major factor in specificity. Similarly, the only obvious charges in the vicinity of the heme, other than the heine proprionates, are the negative charge provided by Cys-75 on one side and the positive charge provided by Arg-4 on the other side. Only the former would seem to be a candidate for repelling cyanide or azide. Obviously, an understanding of the structure of the CO-bound form of CooA will be necessary to further clarify the basis for CO specificity. The mere fact that wild-type CooA does not respond to other effectors does not preclude the the possibility of CooA variants with altered specificity. For example, CooA variants that lack His-77, a normal ligand in the reduced form, are now capable of binding cyanide, while retaining their ability to bind CO. H77Y also shows an extremely low level of activation by cyanide in the very sensitive in vivo assay, although efforts to detect this low activity in vitro have been unsuccessful (71). Nevertheless, the results are encouraging that the combination of targeted mutagenesis and phenotypic screens will allow the identification of CooA variants with altered specificity, which will provide important information concerning the molecular basis for ligand specificity.

VIII. Cooperativily of Ugand Binding It has been difficult to determine the precise cooperativity of wild-type CooA in CO binding: The very low solubility of CO combined with the high affinity of CO for CooA makes the analysis of free CO, and hence the precise nature of cooperativity, technically difficult. However, our present analysis of the data is most consistent with the hypothesis that wild-type CooA is positively cooperative for binding CO, and there is no doubt that CooA variants

CooA:A CO-SENSING TRANSCRIPTIONALFACTOR

55

altered at position 77 are perturbed in that cooperativity (M. V. Thorsteinsson et al., unpublished data). The molecular basis for the implied communication between the two heme molecules has not been identified, but the examination of the crystal structure (Fig. 2A) suggests that the likely pathway involves the lower portions of the C helices of the two protein monomers, which are adjacent to and between the hemes. It seems to be a reasonable hypothesis that CO binding to one heme would affect its positioning relative to the C helix of the other monomer and that this perturbation of the C helix could be transmitted to the heme of the other monomer. While this general scheme is very much like that proposed for activation of CooA by CO, there must be some important differences for the following reason. CooA variants altered at position 77, and therefore lacking the normal His-77 ligand to the reduced form, are still able to bind CO, 'although the degree of cooperativity and CO affinity depends on the nature of the residue at position 77. In addition, these variants all show no detectable activation by CO, demonstrating that activation and cooperativity are distinct. Cooperative binding of CO is not sufficient to support activation and it remains unclear whether or not it is necessary. CooA variants altered at position 77 are also able to hind cyanide and the majority are generally able to do so with positive cooperativity (74). Interestingly, there is no clear pattern among the position 77 variants in terms of their degree of cooperativity in binding either CO and cyanide (74; M. V. Thorsteinsson et al., unpublished data). This indicates that the precise degree of cooperativity is a function of both the small molecule and the residue at position 77. Although the exact mechanism of cooperative CO binding by CooA remains to be determined, it is useful to compare the situation to that of CRE There has been some debate on the matter, but it is generally agreed that CRP is negatively cooperative for binding its effector, cAMP, under physiological conditions (60, 61). Interestingly, the form of CRP with a single molecule of cAMP bound has a higher affinity for DNA, suggesting that it is the physiologically significant form (60). The physiological significance of CRP with two cAMP molecules bound is unclear. We currently do not know if CooA with a single CO molecule bound is active in DNA binding. Either positive cooperativity or activation by a single effector molecule is easy to rationalize for a system that is maximized for sensitivity to the effector. It is more difficult to understand a role for negative cooperativity in this sort of sensing system, unless the doubly bound form is coincidentally less active and the cooperativity is simply a mechanism to reduce the likelihood of that species being present. As the mechanisms of cooperativity in both proteins is unraveled, the comparison between the two homologs, with effector responses of opposite cooperativity, will certainly be informative.

56

GARYE ROBERTSETAL.

IX. Transcriptional Activation by CooA The transcription start sites, as well as the sites of CooA binding, have been determined for the two naturally occurring CooA-regulated promoters in R. rubrum (32, 83). The relative positioning of the sites makes it clear that these are analogous to the Class II sites used by CRP in which the CRP binding site overlaps with the - 3 5 region to stabilize RNA polymerase (RNAP) binding (Fig. 4). Although the nature of the RNAP that CooA utilizes in R. rubrum is unknown, CooA has been heterologously expressed in both Rhodobacter sphaeroides and E. coli, where it supports redox- and CO-dependent gene expression from one of its natural promoters (82). Purified CooA has also been shown to function in in vitro transcription assays with purified Ecr 7° ofE. coli, as well as the RNAP with the major housekeeping sigma from R. sphaeroides (82), which has allowed significant analysis of its behavior, albeit in a heterologous system. While the results might not represent the function of CooA in its normal host, R. rubrum, they certainly indicate the capabilities of this CooA in transcription activation. In these analyses, CooA was shown to be necessary and sufficient for redox- and CO-dependent transcription. This activation required the carboxyl terminal domain of the ot subunit of RNAP for transcription activation. Footprinting analysis showed that CooA facilitated the DNA binding of normal E~r7°, but not that of Ecr 7° lacking the carboxyl terminus of the ~ subunit. An analysis of the residues of the ot subunit involved in interaction with CooA suggested that they are similar, but not identical, to the residues that interact with CRP at Class II promoters (82, 84, 85). It therefore appears that despite the relatively low level of identity between CooA and CRP, CooA behaves rather similarly to CRP in its interaction with RNA polymerase at a Class II promoter in this heterologous system.

FIG. 4. AcartoonofCooAbindingatoneofits twonaturalpromotersinR, rubrum, aCTD and c~NTD refer to the carboxyl and amino domains of the ~ subunit of RNA polymerase, respectively. AR1 is the surface of CooA that interacts with the ~CTD, AR2 interacts with c~NTD, and AR3 interacts with the ~r subunit. Adapted from S. Busby and R. H. Ebright, J. Mol. Biol. 292, 199-213 (1999) by permission of the publisher Academic Press London.

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

57

Another aspect of transcription activation is the nature and role of the regions on the transcriptional activators that interact with polymerase; these are termed "activating regions," and abbreviated AR (Fig. 3). The AR regions have been defined through a variety of analyses of CRP (86) and FNR (87), which have generally similar but not identical properties in terms of interaction with RNA polymerase. For Class II promoters, where the activator protein binds immediately adjacent to the RNA polymerase, AR1 is found on the upstream monomer and interacts with the C-terminal domain of the ot subunit of RNAP, which extends away from the rest of RNAP and reaches around the activator, to contact both the activator and, typically, the adjacent DNA (Fig. 4); this interaction increases the initial binding of RNAP to DNA. In CRP, AR1 is represented by residues 155-164 (the region between the D and E helices in Fig. 3), which lie in the DNA-binding domain; in FNR, this region, together with a nearby region of the effector-binding domain, has been implicated in this type of interaction (87, 88). AR2 is found on the downstream monomer, near the bottom of the effector-binding domain, and interacts with the N-terminal domain of the ot subunit of RNAP to facilitate the isomerization from the closed to the open complex (86); it is unclear if AR2 is functional in FNR. Finally, AR3, which falls at the tip of the 4/5 loop (Fig. 3), is defined as a region that interacts with ~r7° and also facilitates isomerization (89). It is functional in wild-type FNR, but apparently not in wild-type CRP, although such a region can be "created" by certain mutations in the vicinity of the tip of the 4/5 loop. Before the structure of CooA was solved, comparison of the primary sequences of CRP and CooA suggested that only AR3 might be conserved (37, 82), although the requirement for the C-terminal domain of ~ noted above was strongly suggestive of the presence of an AR1 region as well (82). The comparison of the three-dimensional structures of CRP and CooA suggests that there are similar residues in roughly similar positions for both AR2 and AR3. Consistent with this view, CooA variants have been found at positions Lys-26, and Thr-97 (AR2, analogous to CRP residues Lys-22 and Lys-101, respectively) and Val-57 (AR3, analogous to CRP residue Lys-52) that are inactive in promoting expression in vivo, yet retain their normal affinity for DNA upon purification, which is the expected phenotype for variants with defects in AR regions (j. LeDuc and G. P. Roberts, unpublished data). The AR1 region of the CooA structure remains poorly defined and neither gain-of-function nor loss-of-function variants have been assigned to that region to date, although the demonstration of a requirement for the C terminus of ot is strong evidence of such a region in CooA. Based on these results, it is our working hypothesis that CooA has regions analogous to all three AR regions of CRP, although the details of their role might well be mechanistically different.

58

GARYE ROBERTS ET AL.

X. DNA Recognition Properties of CooA The D N A sequence that is bound by CooA is represented by the four "half-sites" found adjacent to the two known CooA-regulated promoters in R. rubrum. Although a "consensus" sequence for CooA binding can be suggested, it is obviously based on a very small data set. In C R P and FNR, the n u m b e r of biologically relevant promoters regulated by each protein has allowed a very good indication of a consensus for each, although natural binding sites rarely come particularly close to those consensus sequences, presumably because such a high-affinity site would interfere with proper regulation (86, 90).

-A-TGTGA

A

...... TCACA-T-

CRP

A-A-TTGAT--A-ATCAAT---

FNR

-A-TGTCA

COOA

...... CGACA-A-

T

B

G'

G184 R180 L

K188~

T

G,A E l 8 1 % o R P / V I

T

-

R185 M~89 ~T

T "..... $212~ L T

c Q178 CooA A _

T18~ ~' 2 ~

G216.f/~

G*.-..E209-~ A

• 8~ \

FNR

AT 1 ~ 1 ~

~)-i L

FIG. 5. (A) CooA, CRP, and FNR "consensus target sequences"; as noted in the text, the CooA target is poorly defined because there are only two known binding sites. (B) Helical wheel presentations of CRP, FNR, and CooA. These are views down the F helix of each protein, with the numbers within the circles indicating the relative position in the helix and the numbers on the outside indicating the specific protein residues. Known or proposed interactions between residues and bases are indicated. Adapted from Ref. 45.

CooA:A CO-SENSINGTRANSCRIPTIONALFACTOR

59

The data in Fig. 5 indicate the "consensus" sites for the three proteins and a "helical wheel" representation of the relative position of the residues in the F helix of each protein. Although there has been some mutagenesis of the F helix of CRP to indicate some of the critical residues for interaction, the "rules" for nucleotide recognition for such a helix have not been worked out (45). Two observations can be made, however. Arg-180 of CRP (at helical position 1) has been proposed to make specific contact with the G in the second position of the CRP consensus (91, 92). Both this residue (Arg-177) and the nucleotide have been conserved in CooA, but neither is present in FNR, consistent with the residue's proposed recognition role. Similarly, the second residue of CRP and FNR has been proposed to be critical for the recognition of a G (the fourth position in the CRP consensus, and the third in the FNR consensus), and CooA has both altered the residue and appears to utilize a C at that position in its consensus (83). As suggested by Fig. 5B, it remains unclear which residues specify several of the highly conserved bases in the CRP and FNR target sequences, and it will be informative to have a more complete analysis of the residues responsible for specific DNA binding in this protein family.

XI. Future Direction and Open Questions The analysis of CooA has already revealed some very interesting insights into important biological questions, such as heme ligation strategies, redox sensing, cooperativity, and the nature of activation in the CRP superfamily of proteins. Nevertheless, a number of open questions remain whose answers will further our understanding of the above issues and address others. (1) What are the structures of oxidized and the CO-bound forms of CooA? The latter structure can be guessed at by analogy with CRP structures, but the former structure, which would provide more details about the ligand switch, is completely unknown. (2) Is the hypothesis about the role of C-helix reorientation upon activation correct? What is the direct cause of the C helix reorientation upon CO binding? Is it positioning of the heme itself? (3) What is the molecular basis for cooperativity in CO binding? (4) Is the form of CooA with a single bound CO molecule active for DNA binding? (5) What is the molecular basis for the specificity of CooA for activation only by CO? Can CooA variants be identified that have dramatic activation in response to cyanide or NO? (6) What are the adventitious ligands that replace Cys-75, His-77, and Pro-2 when these ligands have been mutationally eliminated? These results would tell us something about the flexibility of the heme region. Elimination of these adventitious ligands would also allow us to draw better conclusions about the importance of the normal ligands. (7) How important are the features that have been proposed to stabilize the effector-free and effector-bound forms of CooA? In other words, the recent crystal structure

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o f C o o A allowed a proposal o f a n u m b e r of different interactions in the hinge region b e t w e e n the two forms o f CooA, but the significance o f these has not b e e n tested. (8) Can we develop a b e t t e r u n d e r s t a n d i n g o f the molecular basis o f the redox poise of C o o A and the role o f the ligand switch in that poise? (9) W h a t are the implications of these various features of C o o A on the response of R. r u b r u m to C O ? T h e bulk o f the analysis o f CooA has b e e n p e r f o r m e d either in E. coli or with purified p r o t e i n in vitro, and it will b e interesting to reexamine selected CooA variants in the normal host at physiologically normal levels o f CooA.

ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison; by support from NIH grant GM53228 (to G. P. R), NIH NRSA Fellowship (to M. V. T.), NSF grant MCB9807798 (to T. L. P.), and USDA Fellowship USDA-98353056549 (to W. N. L.). The authors thank Hwan Youn, Yiping He, Mary Chamberlain Conrad, and Jason LeDuc for permission to include unpublished data. Hwan Youn, Kerridwen McNamara, Mary Chamberlain Conrad, and Jason LeDuc also provided helpful suggestions and comments on the text.

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