Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.     

Crystallographic and solution studies of an activation loop mutant of the insulin receptor tyrosine kinase: insights into kinase mechanism

The tyrosine kinase domain of the insulin receptor is subject to autoinhibition in the unphosphorylated basal state via steric interactions involving the activation loop. A mutation in the activation loop designed to relieve autoinhibition, Asp-1161 --> Ala, substantially increases the ability of the unphosphorylated kinase to bind ATP. The crystal structure of this mutant in complex with an ATP analog has been determined at 2.4-A resolution. The structure shows that the active site is unobstructed, but the end of the activation loop is disordered and therefore the binding site for peptide substrates is not fully formed. In addition, Phe-1151 of the protein kinase-conserved DFG motif, at the beginning of the activation loop, hinders closure of the catalytic cleft and proper positioning of alpha-helix C for catalysis. These results, together with viscometric kinetic measurements, suggest that peptide substrate binding induces a reconfiguration of the unphosphorylated activation loop prior to the catalytic step. The crystallographic and solution studies provide new insights into the mechanism by which the activation loop controls phosphoryl transfer as catalyzed by the insulin receptor.     

Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming.

Signaling by insulin requires autophosphorylation of the insulin receptor kinase (IRK) at Tyr1158, Tyr1162, and Tyr1163. Earlier experiments with (32)P-gamma-ATP indicated that the nonphosphorylated IRK (IRK-0P) is relatively inactive, and crystallographic data indicated that the ATP binding site of IRK-0P is blocked by its activation loop. We now show that phosphocreatine (PCr) in combination with hydrogen peroxide serves as an alternative phosphate donor and that ATP and PCr use distinct binding sites. Whereas phosphorylation of the IRK by ATP is inhibited by the nonhydrolyzable competitor adenylyl-imidodiphosphate, phosphorylation by PCr is enhanced. The IRK mutant Tyr1158Phe showed no phosphorylation with PCr but almost normal phosphorylation with ATP, whereas Tyr1162Phe was phosphorylated well with PCr but less then normal with ATP. 3-Dimensional models of IRK-0P revealed that the conversion of any of the four cysteine residues 1056, 1138, 1234, and 1245 into sulfenic acid produces structural changes that bring Tyr1158 into close contact with Asp1083 and render the well-known catalytic site at Asp1132 and Tyr1162 accessible from a direction that differs from the known ATP binding site. The mutant Cys1138Ala, in contrast, showed relatively inaccessible catalytic sites and weak catalytic activity in functional experiments. Taken together, these findings indicate that 'redox priming' of the IRK facilitates its autophosphorylation by PCr in the activation loop.     

Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.     

Structural insights into the autoactivation mechanism of p21-activated protein kinase

p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.     

Conformational changes in the activation loop of the insulin receptor's kinase domain.

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4-0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses > or =90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.     

Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor

The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.     

Role of the activation loop tyrosines in regulation of the insulin-like growth factor I receptor-tyrosine kinase

The tyrosine kinase activity of insulin-like growth factor I receptor (IGF1R) is under tight control. Ligand binding to the extracellular portion of IGF1R stimulates autophosphorylation at three sites (Tyr1131, Tyr1135, and Tyr1136) in the activation loop within the tyrosine kinase catalytic domain. Autophosphorylation at all three sites is required for maximum enzyme activity, and for IGF1-stimulated cellular activity of the receptor. Previous studies have not clarified the contributions of the individual tyrosines to enzymatic activation. Here, we produced single Tyr-to-Phe mutations at these positions, and compared activities of the purified kinase domains (unphosphorylated and phosphorylated) with wild-type IGF1R. Rates of autophosphorylation of the three mutants were more rapid than for wild-type IGF1R; this was most apparent for the Y1135F mutant. Substrate phosphorylation studies on the unphosphorylated forms of IGF1R confirmed that the value of Vmax for Y1135F was elevated relative to wild-type IGF1R, consistent with a disruption of an autoinhibitory interaction. In contrast, activity measurements on the fully phosphorylated enzymes indicated that kcat/Km values were lowered relative to wild-type IGF1R; this effect was most dramatic for Y1136F. We confirmed these findings using limited proteolysis and tryptophan fluorescence experiments. The results demonstrate that Tyr1135 plays a particularly important role in stabilizing the autoinhibited conformation of the activation loop, while Tyr1136 plays the key role in stabilizing the open, activated conformation of IGF1R.     

A novel mode of Gleevec binding is revealed by the structure of spleen tyrosine kinase

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.     

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Br?nsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.     

Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase

In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.     

Asp-120 locates Zn2 for optimal metallo-beta-lactamase activity

Metallo-beta-lactamases are zinc-dependent hydrolases that inactivate beta-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-beta-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the beta-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule.     

Autoregulation by the juxtamembrane region of the human ephrin receptor tyrosine kinase A3 (EphA3)

Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamembrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.     

Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.     

Autophosphorylation Is a Mechanism of Inhibition in Twitchin Kinase

Titin-like kinases are muscle-specific kinases that regulate mechanical sensing in the sarcomere. Twitchin kinase (TwcK) is the best-characterized member of this family, both structurally and enzymatically. TwcK activity is auto-inhibited by a dual intrasteric mechanism, in which N- and C-terminal tail extensions wrap around the kinase domain, blocking the hinge region, the ATP binding pocket and the peptide substrate binding groove. Physiologically, kinase activation is thought to occur by a stretch-induced displacement of the inhibitory tails from the kinase domain. Here, we now show that TwcK inhibits its catalysis even in the absence of regulatory tails, by undergoing auto-phosphorylation at mechanistically important elements of the kinase fold. Using mass spectrometry, site-directed mutagenesis and catalytic assays on recombinant samples, we identify residues T212, T301, T316 and T401 as primary auto-phosphorylation sites in TwcK in vitro. Taken together, our results suggest that residue T316, located in the peptide substrate binding P + 1 loop, is the dominantly regulatory site in TwcK. Based on these findings, we conclude that TwcK is regulated through a triple-inhibitory mechanism consisting of phosphorylation and intrasteric blockage, which is responsive not only to mechanical cues but also to biochemical modulation. This implies that mechanically stretched conformations of TwcK do not necessarily correspond to catalytically active states, as previously postulated. This further suggests a phosphorylation-dependent desensitization of the TwcK-mediated mechanoresponse of the sarcomere in vivo.     

Functional contribution of a conserved, mobile loop histidine of phosphoribulokinase

In the Rhodobacter sphaeroides phosphoribulokinase (PRK) structure, there are several disordered regions, including a loop containing invariant residues Y98 and H100. The functional importance of these residues has been unclear. PRK is inactivated by diethyl pyrocarbonate (DEPC) and protected by the substrates ATP and Ru5P, as well as by the competitive inhibitor, 6-phosphogluconate, suggesting active site histidine residue(s). PRK contains only three invariant histidines: H45, H100, and H134. Previous mutagenesis studies discount significant function for H134, but implicate H45 in Ru5P binding. PRK mutant H45N is inactivated by DEPC, implicating a second active site histidine. To evaluate the function of H100, as well as another invariant loop residue Y98, PRK mutants Y98L, H100A, H100N, and H100Q were characterized. Mutant PRK binding stoichiometries for the fluorescent alternative substrate, trinitrophenyl-ATP, as well as the allosteric activator, NADH, are comparable to wild-type PRK values, suggesting intact effector and substrate binding sites. The K(mRu5P) for the H100 mutants shows modest eight- to 14-fold inflation effects, whereas Y98L exhibits a 40-fold inflation for K(mRu5P). However, Y98L's K(i) for the competitive inhibitor 6-phosphogluconate is close to that of wild-type PRK. These observations suggest that Y98 and H100 are not essential Ru5P binding determinants. The Vm of Y98L is diminished 27-fold compared with wild-type PRK. In contrast, H100A, H100N, and H100Q exhibit significant decreases in Vm of 2600-, 2300-, and 735-fold, respectively. Results suggest that the mobile region containing Y98 and H100 must contribute to PRK's active site. Moreover, H100's imidazole significantly influences catalytic efficiency.     

The third conformation of p38α MAP kinase observed in phosphorylated p38α and in solution

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T?GY?/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T?GY?) and ERK2 (ERK2/T?EY?). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.     

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.     

Effects of mutations of active site residues and amino acids interacting with the Ω loop on substrate activation of butyrylcholinesterase

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Ω loop. To determine whether the Ω loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10 000-fold, indicating severe alteration in the binding area in W430A. The k_cat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Ω loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.     

The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.