Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1)

Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 μM. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.     

Characterization of mitogen-activated protein kinase (MAPK) dimers

Phosphorylated ERK2 has an increased capacity to form homodimers relative to unphosphorylated ERK2. We have characterized the nature of the ERK2 dimer and have mutated residues in the crystal dimer interface to examine the impact of dimerization on ERK2 activity. Analysis of the mutants by gel filtration indicates that at least five residues must be mutated simultaneously to produce an ERK2 mutant that is predominantly monomeric. Mutants, whether monomers or dimers, have specific protein kinase activities under fixed assay conditions that are roughly equivalent to wild-type ERK2. The ratio of dimers to monomers is increased as the salt concentration increases, consistent with a strong hydrophobic contribution to the energy of dimer formation. ERK2 dimerization also requires divalent cations. Sedimentation analysis indicates that the related c-Jun N-terminal kinase SAPKalphaI/JNK2 also forms dimers, but dimerization displays no dependence on phosphorylation; the unphosphorylated and phosphorylated forms of the kinase behave similarly, with low micromolar dimer dissociation constants.     

Receptor-like protein tyrosine phosphatase alpha homodimerizes on the cell surface

We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase alpha (RPTPalpha) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTPsigma D1 catalytic domain. Most recently, we have also shown that enforced dimerization inhibits the biological activity of full-length RPTPalpha in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTPalpha dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTPalpha exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTPalpha biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTPalpha homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization. Finally, we also provide evidence that both the RPTPalpha extracellular domain and the transmembrane domain were independently able to homodimerize. These results lead us to propose a zipper model in which inactive RPTPalpha dimers are stabilized by multiple, relatively weak dimerization interfaces. Dimerization in this manner would provide a potential mechanism for negative regulation of RPTPalpha. Such RPTPalpha dimers could be activated by extracellular ligands or intracellular binding proteins that induce monomerization or by intracellular signaling events that induce an open conformation of the dimer.     

Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation.

IL-8 dimers have been observed in NMR and X-ray structures of the protein. We have engineered IL-8 monomers by mutations of residues throughout the dimer interface, which introduce hindrance determinants to dimerization. These IL-8 variants are shown by NMR to have wild-type monomer folding, but by ultracentrifugation to have a range of dimerization constants from microM to mM, as compared with a dimerization constant of about 10 microM for wild-type IL-8, under physiological salt and temperature conditions. The monomeric variants of IL-8 bind the erythrocyte chemokine receptor DARC, as well as the neutrophil IL-8 receptors CXCR1 and CXCR2 with affinities similar to that of wild-type IL-8. In addition, the monomeric variants were shown to have agonist activity, with similar potency to wild-type, in both Ca(2+)-flux assays on CXCR1 and CXCR2 transfected cells, and in chemotaxis assays on neutrophils. Thus, these variants confirm that monomeric IL-8 is functionally equivalent to wild-type in vitro assays. We have also investigated the effects of various solution conditions upon IL-8 dimer formation using analytical ultracentrifugation. At salt concentrations, temperatures, and pH conditions lower than physiological, the dimerization affinity of IL-8 is greatly enhanced. This suggests that, under some conditions, IL-8 dimer formation may occur at concentrations of IL-8 considerably lower than 10 microM, with consequences in vivo that are yet to be determined.     

Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics.

Dialysis kinetics measurements have been made to study the effect of ionic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals. Using glutaraldehyde cross-linked dimers of lysozyme, we have determined that both monomers and dimers can escape from 25,000 molecular weight cutoff dialysis membranes with velocity constants of 5.1 x 10(-7) and 1.0 x 10(-7) s(-1) for the monomer and dimer species, respectively. The flux from 25K MWCO membranes has been measured for lysozyme in pH 4.0 buffered solutions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concentrations. Assuming that dimerization is the first step in crystallization, a simple monomer to dimer equilibrium was used to model the flux rates. Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respectively. These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions. At high protein concentrations and high supersaturation, the dimerization model does not describe the data well. However, the Li model that uses a pathway of monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer fits the measured flux data remarkably well suggesting the presence of higher order aggregates in crystallizing solutions.     

Importance of the N terminus of rous sarcoma virus protease for structure and enzymatic function

All retrovirus proteases (PRs) are homodimers, and dimerization is essential for enzymatic function. The dimer is held together largely by a short four-stranded antiparallel beta sheet composed of the four or five N-terminal amino acid residues and a similar stretch of residues from the C terminus. We have found that the enzymatic and structural properties of Rous sarcoma virus (RSV) PR are exquisitely sensitive to mutations at the N terminus. Deletion of one or three residues, addition of one residue, or substitution of alanine for the N-terminal leucine reduced enzymatic activity on peptide and protein substrates 100- to 1,000-fold. The purified mutant proteins remained monomeric up to a concentration of about 2 mg/ml, as determined by dynamic light scattering. At higher concentrations, dimerization was observed, but the dimer lacked or was deficient in enzymatic activity and thus was inferred to be structurally distinct from a wild-type dimer. The mutant protein lacking three N-terminal residues (DeltaLAM), a form of PR occurring naturally in virions, was examined by nuclear magnetic resonance spectroscopy and found to be folded at concentrations where it was monomeric. This result stands in contrast to the report that a similarly engineered monomeric PR of human immunodeficiency virus type 1 is unstructured. Heteronuclear single quantum coherence spectra of the mutant at concentrations where either monomers or dimers prevail were nearly identical. However, these spectra differed from that of the dimeric wild-type RSV PR. These results imply that the chemical environment of many of the amide protons differed and thus that the three-dimensional structure of the DeltaLAM PR mutant is different from that of the wild-type PR. The structure of this mutant protein may serve as a model for the structure of the PR domain of the Gag polyprotein and may thus give clues to the initiation of proteolytic maturation in retroviruses.     

Complete crystal structure of monocyte chemotactic protein-2, a CC chemokine that interacts with multiple receptors

Monocyte chemotactic protein 2 (MCP-2) is a CC chemokine that utilizes multiple cellular receptors to attract and activate human leukocytes. MCP-2 is a potent inhibitor of HIV-1 by virtue of its high-affinity binding to the receptor CCR5, one of the major coreceptors for HIV-1. Although a few structures of CC chemokines have been reported, none of these was determined with the N-terminal pyroglutamic acid residue (pGlu1) and a complete C-terminus. pGlu1 is essential for the chemotactic activity of MCP-2. Recombinant MCP-2 has Gln1 at the N terminus, 12-15% of which cyclizes automatically and forms pGlu1. The chemotactic activity of such MCP-2 mixture, which contains 12-15% pGlu1-form and 85-88% Gln1-form protein, is approximately 10 times lower when compared with that of fully cyclized MCP-2 preparation. Therefore, this chemokine is practically inactive without pGlu1. We have determined the complete crystal structure of MCP-2 that contains both pGlu1 and an intact C-terminus. With the existence of pGlu1, the conformation of the N-terminus allows two additional interactions between the two subunits of MCP-2 dimer: a hydrogen bond between pGlu1 and Asn17 and a salt bridge between Asp3 and Arg18. Consequently, both pGlu1 are anchored and buried, and thereby, both N-terminal regions are protected against protease degradation. We have also observed not previously reported extended helical nature of the C terminal region, which covers residues 58-74.     

Chemical modification of a variant of human MIP-1alpha; implications for dimer structure

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.     

Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2.CCL13, CCL2.CCL11 and CCL8.CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8.CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8.CCL11.Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.     

Dimer dissociation is essential for interleukin-8 (IL-8) binding to CXCR1 receptor

Chemokines play a fundamental role in trafficking of immune cells and in host defense against infection. The role of chemokines in the recruitment process is highly regulated spatially and temporally and involves interactions with G protein-coupled receptors and cell surface glycosaminoglycans. The dynamic equilibrium between chemokine monomers and dimers, both free in solution and in cell surface-bound forms, regulates different components of recruitment such as chemotaxis and receptor signaling. The binding and activity of the chemokine interleukin-8 (IL-8) for its receptors, previously studied using "trapped" non-associating monomers and non-dissociating dimers, show that the monomer has a native-like function but support conflicting roles for the dimer. We have measured the binding of native IL-8 to the CXCR1 N-domain, using isothermal titration calorimetry and sedimentation equilibrium techniques. The N-domain constitutes a critical binding site, and IL-8 binding affinity to the receptor N-domain is in the same concentration range as the IL-8 monomerdimer equilibrium. We observed that only the IL-8 monomer, and not the dimer, is competent in binding the receptor N-domain. Based on our results, we propose that IL-8 dimerization functions as a negative regulator for the receptor function and as a positive regulator for binding to glycosaminoglycans and that both play a role in the neutrophil recruitment process.     

NMR structure of CXCR3 binding chemokine CXCL11 (ITAC)

CXCL11 (ITAC) is one of three chemokines known to bind the receptor CXCR3, the two others being CXCL9 (Mig) and CXCL10 (IP-10). CXCL11 differs from the other CXCR3 ligands in both the strength and the particularities of its receptor interactions: It has a higher affinity, is a stronger agonist, and behaves differently when critical N-terminal residues are deleted. The structure of CXCL11 was determined using solution NMR to allow comparison with that of CXCL10 and help elucidate the source of the differences. CXCL11 takes on the canonical chemokine fold but exhibits greater conformational flexibility than has been observed for related chemokines under the same sample conditions. Unlike related chemokines such as IP-10 and IL-8, ITAC does not appear to form dimers at millimolar concentrations. The origin for this behavior can be found in the solution structure, which indicates a beta-bulge in beta-strand 1 that distorts the dimerization interface used by other CXC chemokines.     

Prolines in βA-sheet of neural cadherin act as a switch to control the dynamics of the equilibrium between monomer and dimer

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of the adhesive partners via swapping of their βA-sheets and docking of tryptophan-2 in the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of calcium favors dimer formation by creating strain in the monomer and lowering the energetic barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherins have two proline residues in the βA-sheet. We focused our studies on the role of these two prolines in calcium-dependent dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very stable dimers that required denaturation of protein to disassemble in the apo conditions. In summary, the proline residues act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.     

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.     

Characterization of the monomer-dimer equilibrium of human cytomegalovirus protease by kinetic methods

Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported Kd values for this dimer (between 0.6 and 6 microM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such Kd values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.     

Flipping the switch from monomeric to dimeric CV-N has little effect on antiviral activity

Cyanovirin-N can exist in solution in monomeric and domain-swapped dimeric forms, with HIV-antiviral activity being reported for both. Here we present results for CV-N variants that form stable solution dimers: the obligate dimer [DeltaQ50]CV-N and the preferential dimer [S52P]CV-N. These variants exhibit comparable DeltaG values (10.6 +/- 0.5 and 9.4 +/- 0.5 kcal.mol(-1), respectively), similar to that of stabilized, monomeric [P51G]CV-N (9.8 +/- 0.5 kcal.mol(-1)), but significantly higher than wild-type CV-N (4.1 +/- 0.2 kcal.mol(-1)). During folding/unfolding, no stably folded monomer was observed under any condition for the obligate dimer [DeltaQ50]CV-N, whereas two monomeric, metastable species were detected for [S52P]CV-N at low concentrations. This is in contrast to our previous results for [P51G]CV-N and wild-type CV-N, for which the dimeric forms were found to be the metastable species. The dimeric mutants exhibit comparable antiviral activity against HIV and Ebola, similar to that of wild-type CV-N and the stabilized [P51G]CV-N variant.     

Activity in monomers of human cytomegalovirus protease.

Herpesvirus proteases require dimerization for activity, although crystallographic data indicate that each monomeric subunit possesses a well-separated and complete active site. This suggests that dimerization stabilizes the monomeric protease subunits in an active conformation. Chemical cross-linking with disuccinimidyl glutarate was used to capture human cytomegalovirus protease in its various conformations. The cross-linked protease retained activity under mildly chaotropic conditions (0.25 to 0.75 M urea) in contrast to non-cross-linked protease which lost activity. Identification of active protease species by incorporation of radioactive diisopropylfluorophosphate showed that in addition to cross-linked dimers, cross-linked protease monomers were responsible for a significant fraction of the total protease activity. These results are consistent with the hypothesis that herpesvirus protease activation occurs by stabilization of an active conformer in the dimer.     

Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line

A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.     

Dimerization of Vaccinia virus VH1 is essential for dephosphorylation of STAT1 at tyrosine 701

The gene product of Vaccinia virus gene H1, VH1, is the first identified dual specificity phosphatase (DSP). The human genome encodes 38 different VH1-like DSPs, which include major regulators of signaling pathways, highly dysregulated in disease states. VH1 down-regulates cellular antiviral response by dephosphorylating activated STAT1 in the IFN-γ/STAT1 signaling pathway. In this report, we have investigated the molecular basis for VH1 catalytic activity. Using small-angle x-ray scattering (SAXS), we determined that VH1 exists in solution as a boomerang-shaped dimer. Targeted alanine mutations in the dimerization domain (aa 1-27) decrease phosphatase activity while leaving the dimer intact. Deletion of the N-terminal dimer swapped helix (aa 1-20) completely abolishes dimerization and severely reduces phosphatase activity. An engineered chimera of VH1 that contains only one active site retains wild-type levels of catalytic activity. Thus, a dimeric quaternary structure, as opposed to two cooperative active sites within the same dimer is essential for VH1 catalytic activity. Together with laforin, VH1 is the second DSP reported in literature for which dimerization via an N-terminal dimerization domain is necessary for optimal catalytic activity. We propose that dimerization may represent a common mechanism to regulate the activity and substrate recognition of DSPs, often assumed to function as monomers.     

Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.