Atomistic Insights into Regulatory Mechanisms of the HER2 Tyrosine Kinase Domain: A Molecular Dynamics Study

HER2 (ErbB2/Neu) is a receptor tyrosine kinase belonging to the epidermal growth factor receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. Although several crystal structures of ErbB kinases have been solved, the precise mechanism of HER2 activation remains unknown, and it has been suggested that HER2 is unique in its requirement for phosphorylation of Y877, a key tyrosine residue located in the activation loop. To elucidate mechanistic details of kinase domain regulation, we performed molecular dynamics simulations of a homology-modeled HER2 kinase structure in active and inactive conformations. Principal component analysis of the atomistic fluctuations reveals a tight coupling between the activation loop and catalytic loop that may contribute to alignment of residues required for catalysis in the active kinase. The free energy perturbation method is also employed to predict a role for phosphorylated Y877 in stabilizing the kinase conformations. Finally, simulation results are presented for a HER2/EGFR heterodimer and reveal that the dimeric interface induces a rearrangement of the αC helix toward the active conformation. Elucidation of the molecular regulatory mechanisms in HER2 will help establish structure-function relationships in the wild-type kinase, as well as predict mutations with a propensity for constitutive activation in HER2-mediated cancers.     

A negative regulatory role for Y1111 on the Tie-2 RTK

Tie2 is a receptor tyrosine kinase (RTK) essential for aspects of both normal and pathological angiogenesis. Understanding how this receptor is regulated is important for development of therapeutic angiogenic agents. Evidence suggests the C-terminal tail of the receptor plays a negative regulatory role in Tie2 signaling and function. Here we investigated the role of a specific C-tail residue, Y1111, in Tie2 signaling by generating a number of receptor point mutants. We found that mutation of this site to phenylalanine (Y1111F) results in an increase in receptor phosphorylation and kinase activity, as well as increased downstream signaling. Furthermore, mutation of Y1111 to the highly charged aspartate (Y1111D) or glutamate (Y1111E) results in even more dramatic increase in receptor phosphorylation and activity. Limited protease digestion studies indicate that these mutations may alter receptor conformation and potentially relieve negative inhibition imparted by the C-tail of Tie2. These studies suggest that Y1111 plays a key role in negative regulation of Tie2 activity and they provide important insight into molecular mechanisms behind the intrinsic ability of this RTK to regulate its own activity.     

Identification of tyrosine 972 as a novel site of Jak2 tyrosine kinase phosphorylation and its role in Jak2 activation

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.     

Expression, purification, and characterization of His20 mutants of rat mevalonate kinase

Mevalonate kinase plays a key role in regulating the biosynthesis of cholesterol in animal cells. Human mevalonate kinase His20Pro has been reported as one of the three common mutations in the mevalonate kinase gene in mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome patients. His20 is also a highly conserved residue among all aligned mevalonate kinase sequences. To study the role of His20 of mevalonate kinase, a variety of mutant expression plasmids of rat mevalonate kinase including pRMK(H20L), pRMK(H20Y), and pRMK(H20K) were constructed using site-directed mutagenesis, and mutant proteins were overexpressed and purified. CD spectroscopy of wild-type protein and mutants indicated that mutations H20L and H20Y did not induce significant secondary structural changes. The results from kinetic studies showed that this highly conserved histidine is an important residue for the function of the enzyme.     

Function of Focal Adhesion Kinase Scaffolding to Mediate Endophilin A2 Phosphorylation Promotes Epithelial-Mesenchymal Transition and Mammary Cancer Stem Cell Activities in Vivo

Tyrosine kinases have been shown to play critical roles in cancer development and progression, and their inhibitors hold the potential as effective targeted therapies for breast and other cancers. However, some of these kinases like focal adhesion kinase (FAK) also possess scaffolding functions in intracellular signaling, but such kinase-independent functions of FAK or other kinases have not been examined in cancer directly in vivo. Here, we report that disruption of the function of FAK scaffolding through its Pro-878/881 motif suppressed mammary tumor growth and metastasis in a well characterized murine model of human breast cancer. P878A/P881A mutation in the endogenous FAK gene decreased the expression of markers for epithelial-mesenchymal transition (EMT) and mammary cancer stem cell (MaCSC) activities in tumors derived from mutant mice. This mutation disrupted the function of FAK scaffolding to mediate endophilin A2 phosphorylation at Tyr-315 by Src, leading to the decreased surface expression of MT1-MMP, as observed previously in transformed fibroblasts in vitro. Inhibition of the downstream components of this FAK scaffolding function by Y315F endophilin A2 mutant or MT1-MMP knockdown reduced markers for EMT and MaCSC activities. Conversely, bypass of the scaffolding function using the phosphorylation mimic mutant Y315E endophilin A2 or endophilin A2 knockdown rescued the decreased markers for EMT and MaCSCs as well as surface expression of MT1-MMP in tumor cells harboring the P878A/P881A mutation. Together, these results identify a novel role of FAK scaffolding function in breast cancer, which could serve as a new target in combination with kinase inhibition for more effective treatment strategies.     

Six X-linked agammaglobulinemia-causing missense mutations in the Src homology 2 domain of Bruton's tyrosine kinase: phosphotyrosine-binding and circular dichroism analysis

Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).     

Structural basis for peptide binding in protein kinase A. Role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop

For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr-197. This phosphate anchors the activation loop in the proper conformation and contributes to catalytic efficiency by enhancing the phosphoryl transfer rate and increasing the affinity for ATP (1). The crystal structure of the catalytic subunit bound to ATP, and the inhibitor peptide, IP20, highlights the contacts made by the Thr-197 phosphate as well as the role adjacent residues play in contacting the substrate peptide. Glu-203 and Tyr-204 interact with arginines in the consensus sequence of PKA substrates at the P-6 and P-2 positions, respectively. To assess the contribution that each residue makes to peptide recognition, the kinetic properties of three mutant proteins (E203A, Y204A, and Y204F) were monitored using multiple peptide substrates. The canonical peptide substrate, Kemptide, as well as a longer 9-residue peptide and corresponding peptides with alanine substitutions at the P-6 and P-2 positions were used. While the effect of Glu-203 is more localized to the P-6 site, Tyr-204 contributes to global peptide recognition. An aromatic hydrophobic residue is essential for optimal peptide recognition and is conserved throughout the protein kinase family.     

Molecular mechanisms of "off-on switch" of activities of human IDH1 by tumor-associated mutation R132H

Human cytosolic NADP-IDH (IDH1) has recently been found to be involved in tumorigenesis. Notably, the tumor-derived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting α-ketoglutarate (αKG) to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate (ICT) bound. The structural data together with mutagenesis and biochemical data reveal a previously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conformation, therefore shedding light on the molecular mechanism of the "off switch" of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained αKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of αKG during the transfer of a hydride anion from NADPH to αKG, which provides new insights into the mechanism of the "on switch" of the hypothetically oncogenic reduction activity of IDH1 by this mutation.     

Role of HER2 gene overexpression in breast carcinoma

The HER2 proto-oncogene encodes a transmembrane glycoprotein of 185 kDa (p185(HER2)) with intrinsic tyrosine kinase activity. Amplification of the HER2 gene and overexpression of its product induce cell transformation. Numerous studies have demonstrated the prognostic relevance of p185(HER2), which is overexpressed in 10% to 40% of human breast tumors. Recent data suggest that p185(HER2) is a ligand orphan receptor that amplifies the signal provided by other receptors of the HER family by heterodimerizing with them. Ligand-dependent activation of HER1, HER3, and HER4 by EGF or heregulin results in heterodimerization and, thereby, HER2 activation. HER2 overexpression is associated with breast cancer patient responsiveness to doxorubicin, to cyclophosphamide, methotrexate, and fluorouracil (CMF), and to paclitaxel, whereas tamoxifen was found to be ineffective and even detrimental in patients with HER2-positive tumors. In vitro analyses have shown that the role of HER2 overexpression in determining the sensitivity of cancer cells to drugs is complex, and molecules involved in its signaling pathway are probably the actual protagonists of the sensitivity to drugs. The association of HER2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor-specific therapies. A number of approaches are being investigated as possible therapeutic strategies that target HER2: (1) growth inhibitory antibodies, which can be used alone or in combination with standard chemotherapeutics; (2) tyrosine kinase inhibitors (TKI), which have been developed in an effort to block receptor activity because phosphorylation is the key event leading to activation and initiation of the signaling pathway; and (3) active immunotherapy, because the HER2 oncoprotein is immunogenic in some breast carcinoma patients.     

Integrated Exploitation of the Structural Diversity Space of Chemotherapy Drugs to Selectively Inhibit HER2 T798M Mutant in Lung Cancer

An acquired T798M gatekeeper mutation in human epidermal growth factor receptor 2 (HER2) kinase can cause drug resistance to anti‐HER2 chemotherapy drugs in lung cancer. Previously, the reversible pan‐kinase inhibitor staurosporine has been found to selectively inhibit the HER2 T798M mutant over wild‐type kinase, suggesting that the staurosporine scaffold is potentially to develop mutant‐selective inhibitors. Here, we systematically evaluated the chemical space of staurosporine scaffold‐based compounds in response to HER2 T798M mutation at structural, energetic and molecular levels by using an integrated analysis strategy. With this strategy, we were able to identify several novel wild‐type sparing inhibitors with high or moderate selectivity, which are comparable to or even better than that of the parent compound staurosporine. Molecular modeling and structural analysis revealed that noncovalent contacts can form between the side chain of mutated residue Met798 and selective inhibitor ligands, which may improve the favorable interaction energy between the kinase and inhibitor and reduce the unfavorable desolvation penalty upon the kinase–inhibitor binding.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

Structure-function relationship in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the tetrahydrofolate (H(4)-folate)-dependent retro-aldol cleavage of serine to form 5,10-methylene H(4)-folate and glycine. The structure-function relationship of SHMT was studied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an 'open' to 'closed' conformation rather than proton abstraction from the hydroxyl group of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate. The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s) complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.     

Conformation and properties of hammerhead-type RNA enzymes

We designed a hammerhead-type RNA enzyme system which consists of three ribooligonucleotide strands (1-3), synthesized these oligomers and their analogues, and examined conformation and properties of the RNA complexes. Imino proton NMR spectra of the complexes were measured and the signals were assigned by comparison with the spectra of some model duplexes. Examination of a complex containing a G----I mutation (Y = I) revealed that the 2-amino group of the third guanosine residue in the loop 1 (L1) plays an important role for maintaining both the activity and loop conformation.     

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.     

The role of water in the catalytic efficiency of triosephosphate isomerase

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.     

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT2A receptor

The 5-HT_2A receptor (5-HT_2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT_2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT_2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT_2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT_2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT_2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT_2AR.     

Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand,hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.     

Regulation of the wild-type and Y1235D mutant Met kinase activation

Met receptor tyrosine kinase plays a crucial role in the regulation of a large number of cellular processes and, when deregulated by overexpression or mutations, leads to tumor growth and invasion. The Y1235D mutation identified in metastases was shown to induce constitutive activation and a motile-invasive phenotype on transduced carcinoma cells. Wild-type Met activation requires phosphorylation of both Y1234 and Y1235 in the activation loop. We mapped the major phosphorylation sites in the kinase domain of a recombinant Met protein and identified the known residues Y1234 and Y1235 as well as a new phosphorylation site at Y1194 in the hinge region. Combining activating and silencing mutations at these sites, we characterized in depth the mechanism of activation of wild-type and mutant Met proteins. We found that the phosphotyrosine mimetic mutation Y1235D is sufficient to confer constitutive kinase activity, which is not influenced by phosphorylation at Y1234. However, the specific activity of this mutant was lower than that observed for fully activated wild-type Met and induced less phosphorylation of Y1349 in the signaling site, indicating that this mutation cannot entirely compensate for a phosphorylated tyrosine at this position. The Y1194F silencing mutation yielded an enzyme that could be activated to a similar extent as the wild type but with significantly slower activation kinetics, underlying the importance of this residue, which is conserved among different tyrosine kinase receptors. Finally, we observed different interactions of wild-type and mutant Met with the inhibitor K252a that may have therapeutic implications for the selective inhibition of this kinase.     

Roles of the heme distal residues of FixL in O2 sensing: a single convergent structure of the heme moiety is relevant to the downregulation of kinase activity

FixL is a heme-based O(2) sensor, in which the autophosphorylation is regulated by the binding of exogenous ligands such as O(2) and CN(-). In this study, mutants of the heme distal Arg200, Arg208, Ile209, Ile210, and Arg214 residues of SmFixL were characterized biochemically and physicochemically, because it has been suggested that they are significant residues in ligand-linked kinase regulation. Measurements of the autoxidation rate, affinities, and kinetics of ligand binding revealed that all of the above residues are involved in stabilization of the O(2)-heme complex of FixL. However, Arg214 was found to be the only residue that is directly relevant to the ligand-dependent regulation of kinase activity. Although the wild type and R214K and R214Q mutants exhibited normal kinase regulation, R214A, R214M, R214H, and R214Y did not. (13)C and (15)N NMR analyses for (13)C(15)N(-) bound to the truncated heme domains of the Arg214 mutants indicated that, in the wild type and the foregoing two mutants, the heme moiety is present in a single conformation, but in the latter four, the conformations fluctuate possibly because of the lack of an interaction between the iron-bound ligand and residue 214. It is likely that such a rigid conformation of the ligand-bound form is important for the downregulation of histidine kinase activity. Furthermore, a comparison of the NMR data between the wild type and R214K and R214Q mutants suggests that a strong electrostatic interaction between residue 214 and the iron-bound ligand is not necessarily required for the single convergent structure and eventually for the downregulation of FixL.