Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase

Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV](n), previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-beta suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.     

Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD

KdpD/KdpE two‐component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C‐terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high‐resolution crystal structure of KdpD GAF domain (KdpD^G) consisting of five α‐helices, four β‐sheets and two large loops. KdpD^G forms a symmetry‐related dimer, wherein parallelly arranged monomers contribute to a four‐helix bundle at the dimer‐interface, SAXS analysis of KdpD C‐terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide‐binding GAF domains to KdpD^G reveals structural differences arising from naturally occurring substitutions in primary sequence of KdpD^G that modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpD^G in dimerization and transmission of signal to the kinase domain.     

High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation

Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.     

Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links,Insertions, or Deletions in the Extracellular Juxtamembrane Region: MECHANISTIC IMPLICATIONS*

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.     

Activation of receptor protein-tyrosine kinases from the cytoplasmic compartment

It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one 'activator' domain activates the other 'receiver' domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.     

Receptor tyrosine kinase transmembrane domains: Function,dimer structure and dimerization energetics

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) play an active role in signaling. They contribute to the stability of full-length receptor dimers and to maintaining a signaling-competent dimeric receptor conformation. In an exciting new development, two structures of RTK TM domains have been solved, a break-through achievement in the field. Here we review these structures, and we discuss recent studies of RTK TM domain dimerization energetics, possible synergies between domains, and the effects of pathogenic RTK TM mutations on structure and dimerization.Key words: transmembrane domain, dimerization thermodynamics, receptor tyrosine kinases, pathogenic mutations, dimer structure     

Dimer interface of transmembrane domains for neu/erbB-2 receptor dimerization and transforming activation: a model revealed by molecular dynamics simulations

The specific point mutation Val-->Glu664 within the transmembrane domain of the neu/erbB-2 receptor is associated with increased receptor dimerization and increased receptor tyrosine kinase activity resulting in malignant transformation of cells. It is well established that Glu and residues in proximity are necessary for receptor dimerization but many studies suggest that other intramembrane constraints, not yet elucidated, are determinant for transformation. In this work, we investigated dimer models both to understand the structural role of the Glu mutation in the transmembrane domain association and to determine helix-helix contacts required for oncogenic transformation. Different types of helix-helix association based on data resulting from Cys mutational studies of the full wild receptor and spectroscopic data of transmembrane neu peptides have been explored by molecular dynamics simulations. The study leads to propose a model for the dimeric association of the transmembrane domains of the oncogenic neu receptor showing left-handed interactions of the two helices stabilized by symmetrical hydrogen bonding interactions involving the Glu side chain on one helix and the facing carbonyl of Ala661 on the second helix. Contacting residues observed in the symmetric interface explain the transforming activity or the non transforming activity of many neu mutants. Moreover the left-handed coiled coil structure is fully consistent with recent results proving the role of rotational linkage of the transmembrane domain with the kinase domain. Comparison between the predicted dimer model and those presumed from experiments strongly suggests helix flexibility in the extracellular juxtamembrane region.     

Redesigning the procaspase‐8 dimer interface for improved dimerization

Caspase‐8 is a cysteine directed aspartate‐specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase‐8 depends on adaptor‐induced dimerization of procaspase‐8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase‐8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase‐3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase‐8 is a monomer while procaspase‐3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase‐8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase‐8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase‐8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase‐8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.     

Role of c-kit/SCF in cause and treatment of gastrointestinal stromal tumors (GIST)

c-Kit encodes for the receptor tyrosine kinase (RTK) and belongs to type III receptor family. This includes platelet derived growth factor (PDGF) alpha and beta and macrophage colony stimulating factor (mCSF) apart from others. Their characteristic features are the presence of five immunologlobulin like domains in the extracellular region and 70-100 residues long kinase insert domain in the cytoplasmic region. The RTKs activate several signaling pathways within the cells leading to cell proliferation, differentiation, migration or metabolic changes. The Kit ligand-stem cell factor (SCF) induces a rapid and complete receptor dimerization resulting in activation by autophosphorylation of the catalytic tyrosine kinase and generation of signal transduction leading to regulation of cell growth. Various mutations in c-kit such as insertions and deletions (without affecting reading frame) and point mutations in the inhibitory juxtamembrane (JM) domain encoded by exon 11 have been reported in gastrointestinal stromal tumors (GISTs). Thus, c-kit signaling is believed to play a role in tumorigenesis. Efforts are being made to control and treat these tumors by blocking kit signaling using Imatinib with varying degrees of success. This review deals with the features of c-kit, its ligand and roles in gastrointestinal stromal tumors.     

Transmembrane interactions in the activation of the Neu receptor tyrosine kinase

The Neu receptor tyrosine kinase is constitutively activated by a single amino acid change in the transmembrane domain of the receptor. The mutation of Val664 to glutamate or glutamine induces receptor dimerization and autophosphorylation of the receptor's intracellular kinase domain. The ability of this single mutation to activate the receptor is sequence-dependent, suggesting that specific helix-helix interactions stabilize the transmembrane dimer. We have determined the local secondary structure and interhelical contacts in the region of position 664 in peptide models of the activated receptor using solid-state rotational resonance and rotational echo double-resonance (REDOR) NMR methods. Intrahelical (13)C rotational resonance distance measurements were made between 1-(13)C-Thr662 and 2-(13)C-Gly665 on peptides corresponding to the wild-type Neu and activated Neu transmembrane sequences containing valine and glutamate at position 664, respectively. We observed similar internuclear distances (4.5 +/- 0.2 A) in both Neu and Neu*, indicating that the region near residue 664 is helical and is not influenced by mutation. Interhelical (15)N...(13)C REDOR measurements between Gln664 side chains on opposing helices were not consistent with hydrogen bonding between the side chain functional groups. However, interhelical rotational resonance measurements between 1-(13)C-Glu664 and 2-(13)C-Gly665 and between 1-(13)C-Gly665 and 2-(13)C-Gly665 demonstrated close contacts (4.3-4.5 A) consistent with the packing of Gly665 in the Neu* dimer interface. These measurements provide structural constraints for modeling the transmembrane dimer and define the rotational orientation of the transmembrane helices in the activated receptor.     

Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.     

Structural basis for UBA-mediated dimerization of c-Cbl ubiquitin ligase

Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-A resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs.     

Agonistic induction of a covalent dimer in a mutant of natriuretic peptide receptor-A documents a juxtamembrane interaction that accompanies receptor activation

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.     

Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF-dependent signaling requires proteolysis from an inactive single-chain precursor into an active alpha/beta-heterodimer. We show that the serine protease-like HGF beta-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed beta-propeller, where the bottom face of blades 2 and 3 binds to the HGF beta-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF beta binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF beta-chains brings two HGF beta:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.     

The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b)

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.     

Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.     

Strong oligomerization behavior of PDGFβ ?receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.     

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1)

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).     

Characterization of the dimerization domain in the FNR transcription factor

The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.