Structural studies of shikimate dehydrogenase from Bacillus anthracis complexed with cofactor NADP

Bacillus anthracis has been employed as an agent of bioterrorism, with high mortality, despite anti-microbial treatment, which strongly indicates the need of new drugs to treat anthrax. Shikimate pathway is a seven step biosynthetic route which generates chorismic acid from phosphoenol pyruvate and erythrose-4-phosphate. Chorismic acid is the major branch point in the synthesis of aromatic amino acids, ubiquinone, and secondary metabolites. The shikimate pathway is essential for many pathological organisms, whereas it is absent in mammals. Therefore, these enzymes are potential targets for the development of nontoxic antimicrobial agents and herbicides and have been submitted to intensive structural studies. The forth enzyme of this pathway is responsible for the conversion of dehydroshikimate to shikimate in the presence of NADP. In order to pave the way for structural and functional efforts toward development of new antimicrobials we describe the molecular modeling of shikimate dehydrogenase from Bacillus anthracis complexed with the cofactor NADP. This study was able to identify the main residues of the NADP binding site responsible for ligand affinities. This structural study can be used in the design of more specific drugs against infectious diseases.     

Structures of shikimate dehydrogenase AroE and its Paralog YdiB. A common structural framework for different activities

Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 A resolution, respectively. Both enzymes display a similar architecture with two alpha/beta domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms.     

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.     

proP-mediated proline transport also plays a role in Escherichia coli osmoregulation.

Shikimate kinase II was purified to near homogeneity from an Escherichia coli strain which overproduced the enzyme. The apparent Km of this isoenzyme for shikimate was 200 microM, and for ATP it was 160 microM. The Km for shikimate is approximately 100-fold lower than the Km of shikimate kinase I, suggesting that shikimate kinase II is the isoenzyme normally functioning in aromatic biosynthesis. Shikimate kinase II is dependent on metal ions for activity.     

Crystal structure of shikimate 5-dehydrogenase (SDH) bound to NADP: insights into function and evolution

The crystal structure of Methanococcus jannaschii shikimate 5-dehydrogenase (MjSDH) bound to the cofactor nicotinamide adenine dinucleotide phosphate (NADP) has been determined at 2.35 A resolution. Shikimate 5-dehydrogenase (SDH) is responsible for NADP-dependent catalysis of the fourth step in shikimate biosynthesis, which is essential for aromatic amino acid metabolism in bacteria, microbial eukaryotes, and plants. The structure of MjSDH is a compact alpha/beta sandwich with two distinct domains, responsible for binding substrate and the NADP cofactor, respectively. A phylogenetically conserved deep cleft on the protein surface corresponds to the enzyme active site. The structure reveals a topologically new domain fold within the N-terminal segment of the polypeptide chain, which binds substrate and supports dimerization. Insights gained from homology modeling and sequence/structure comparisons suggest that the SDHs represent a unique class of dehydrogenases. The structure provides a framework for further investigation to discover and develop novel inhibitors targeting this essential enzyme.     

Structural and mechanistic analysis of a novel class of shikimate dehydrogenases: evidence for a conserved catalytic mechanism in the shikimate dehydrogenase family

Shikimate dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroshikimate to shikimate. This reaction represents the fourth step of the shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH homologues. Two distinct biochemical roles have been reported for this Lys-Asp pair: as binding residues in YdiB and as a catalytic dyad in AroE and SdhL. Here, we establish that the residues function as a catalytic dyad in Ael1 and, interestingly, in at least one YdiB homologue. The conservation of three-dimensional fold, active site architecture, and catalytic mechanism among members of the SDH family will facilitate the design of drugs targeting the shikimate pathway.     

Structure of the type III pantothenate kinase from Bacillus anthracis at 2.0 A resolution: implications for coenzyme A-dependent redox biology

Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-89]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 A, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.     

Discovery of Helicobacter pylori shikimate kinase inhibitors: bioassay and molecular modeling

Shikimate kinase (SK) is the fifth enzyme in the shikimate pathway and catalyzes the phosphate transfer from ATP to shikimate in generating shikimate 3-phosphate and ADP. SK has been developed as a promising target for the discovery of antibacterial agents. In this report, two small molecular inhibitors (compound 1, 3-methoxy-4-{[2-({2-methoxy-4-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]phenoxy}methyl)benzyl]oxy}benzaldehyde; compound 2, 5-bromo-2-(5-{[1-(3,4-dichlorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}-2-furyl)benzoic acid) against Helicobacter pylori SK (HpSK) were successfully identified with IC(50) values of 5.5+/-1.2 and 6.4+/-0.4 microM, respectively. The inhibition kinetics shows that compound 1 is a noncompetitive inhibitor with respect to both shikimate and MgATP, and compound 2 is a competitive inhibitor toward shikimate and noncompetitive inhibitor with respect to MgATP. The surface plasmon resonance (SPR) technology based analysis reveals that the equilibrium dissociation constants (K(D)s) of compounds 1 and 2 with HpSK enzyme are 4.39 and 3.74 microM, respectively. The molecular modeling and docking of two inhibitors with HpSK reveals that the active site of HpSK is rather roomy and deep, forming an L-shape channel on the surface of the protein, and compound 1 prefers the corner area of L-shape channel, while compound 2 binds the short arm of the channel of SK in the binding interactions. It is expected that our current work might supply useful information for the development of novel SK inhibitors.     

Structural basis for shikimate-binding specificity of Helicobacter pylori shikimate kinase

Shikimate kinase (EC 2.7.1.71) catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP. As the fifth key step in the shikimate pathway for aromatic amino acid biosynthesis in bacteria, fungi, and plants, but not mammals, shikimate kinase represents an attractive target for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here, we report the 1.8-Angstroms crystal structure of Helicobacter pylori shikimate kinase (HpSK). The crystal structure shows a three-layer alpha/beta fold consisting of a central sheet of five parallel beta-strands flanked by seven alpha-helices. An HpSK-shikimate-PO(4) complex was also determined and refined to 2.3 Angstroms, revealing induced-fit movement from an open to a closed form on substrate binding. Shikimate is located above a short 3(10) helix formed by a strictly conserved motif (GGGXV) after beta(3). Moreover, several highly conserved charged residues including Asp33 (in a conserved DT/SD motif), Arg57, and Arg132 (interacting with shikimate) are identified, guiding the development of novel inhibitors of shikimate kinase.     

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.     

High-resolution structure of shikimate dehydrogenase from Thermotoga maritima reveals a tightly closed conformation

Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 Å by molecular replacement. SDH from T. maritima showed a monomeric architecture. The overall structure of SDH from T. maritima comprises the N-terminal α/β sandwich domain for substrate binding and the C-terminal domain for NADP binding. When the T. maritima SDH structure was compared with those of the SDHs from other species, the SDH from T. maritima was in a tightly closed conformation, which should be open for catalysis. Notably, α7 moves toward the active site (∼5 Å), which forces the SDH of T. maritima in a more closed form. Four ammonium sulfate (AMS) ions were identified in the structure. They were located in the active site and appeared to mimic the role of the substrate in terms of the enzyme activity and stability. The new high resolution structural information reported in this study, including the AMS binding sites as a potent inhibitor binding site of SDHs, is expected to supplement the existing structural data and will be useful for structure-based antibacterial discovery against SDHs.     

Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis.

In Bacillus subtilis shikimate kinase enzyme activity can be demonstrated when a small polypeptide forms a trifunctional complex with the bifunctional enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase-chorismate mutase. The shikimate kinase polypeptide whoch carries the catalytic site has been purified to homogeneity by a five-step procedure. The skikimate kinase was determined to have a molecular weight of 10,000 by superfine Sephadex G-75 thin layer chromatography and by calculation of the minimum chemical molecular weight from its amino acid composition. This number corresponds closely to the molecular weight determined by the mobility of the protein following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The enzyme aggregates with itself forming larger molecular weight proteins. Thes aggregational pattersn depend on protein concentration and sulfhydryl bridges. The enzyme activity is completely inhibited by EDTA and the requirement for Mg2+ can be partially replaced by Mn2+, Ca2+, and Co2+. The inhibition of shikimate kinase activity by p-hydroxymercuribenzoate is reversed completely when the enzyme complex is treated with dithiothreitol, suggesting the sulfhydryl groups may be involved with the active site. The trifunctional complex is relatively unstable, and the nonidentical subunits dissociate readily. This dissociation results in a 99% loss in shikimate kinase activity and a 30% decrease in the chorismate mutase-DAHP synthetase activities. Shikimate kinase activity is subject to a variety of controls. It is inhibited by the allosteric effectors chorismate and prephenate, the products of the reaction, ADP, and shikimate 5-phosphate. The activity responds to changes in the energy charge of the cell. Because of the variety of controls exerted on this enzyme, this member of the regulatory complex may represent the key enzyme in the allosteric control of the synthesis of the common precursors of aromatic acid synthesis.     

Shikimate kinase from spinach chloroplasts : purification, characterization, and regulatory function in aromatic amino Acid biosynthesis

Shikimate kinase was purified to near homogenity from spinach Spinacia oleracea L. chloroplasts and found to consist of a single 31 kilodalton polypeptide. The purified enzyme was unstable, but could be stabilized by a variety of added proteins, including oxidized and reduced thioredoxins. Whereas the isolated enzyme was stimulated by mono- and dithiol reagents, the enzyme in intact chloroplasts was unaffected by added thiols and showed only minor response to dark/light transitions. These results indicate that the previously reported stimulation of shikimate kinase activity by reduced thioredoxins is due to enzyme stabilization rather than to activation. In the current study, the purified enzyme was inhibited by added ADP and showed a strong response to energy charge. When intact chloroplasts were incubated in the dark in presence of shikimate, phosphoenolpyruvate and a source of ATP (dihydroxyacetone phosphate or ATP itself under appropriate conditions), aromatic amino acids were formed: phenylalanine and tyrosine. The data indicate that energy charge plays a role in regulating shikimate kinase, thereby controlling the shikimate pathway. An unidentified enzyme of the latter part of the pathway, leading from shikimate-3-phosphate to phenylalanine, appears to be activated by light.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

The 2.3-A crystal structure of the shikimate 5-dehydrogenase orthologue YdiB from Escherichia coli suggests a novel catalytic environment for an NAD-dependent dehydrogenase

We present here the 2.3-A crystal structure of the Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase. This enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been promoted as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the protomer contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue in close apposition to the nicotinamide ring and a clamp over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation in all orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction.     

In silico docking and molecular dynamics simulation of 3-dehydroquinate synthase (DHQS) from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The shikimate pathway is as an attractive target because it is present in bacteria, algae, fungi, and plants but does not occur in mammals. In Mycobacterium tuberculosis (MTB), the shikimate pathway is integral to the biosynthesis of naphthoquinones, menaquinones, and mycobactin. In these study, novel inhibitors of 3-dehydroquinate synthase (DHQS), an enzyme that catalyzes the second step of the shikimate pathway in MTB, were determined. 12,165 compounds were selected from two public databases through virtual screening and molecular docking analysis using PyRx 8.0 and Autodock 4.2, respectively. A total of 18 compounds with the best binding energies (?13.23 to ?8.22 kcal/mol) were then selected and screened for absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, and nine of those compounds were found to satisfy all of the ADME and toxicity criteria. Among those nine, the three compounds—ZINC633887?(binding energy =??10.29 kcal/mol), ZINC08983432?(?9.34 kcal/mol), and PubChem73393?(?8.61 kcal/mol)—with the best binding energies were further selected for molecular dynamics (MD) simulation analysis. The results of the 50-ns MD simulations showed that the two compounds ZINC633887 and PubChem73393 formed stable complexes with DHQS and that the structures of those two ligands remained largely unchanged at the ligand-binding site during the simulations. These two compounds identified through docking and MD simulation are potential candidates for the treatment of TB, and should undergo validation in vivo and in vitro.     

Modulation of phosphoenolpyruvate synthase expression increases shikimate pathway product yields in E. coli

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed-batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3-dehydroshikimic acid. Shikimate pathway byproducts 3-deoxy-D-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3-dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3-dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.     

Structures of Helicobacter pylori shikimate kinase reveal a selective inhibitor-induced-fit mechanism

Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure–activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK·SO₄, R57A, and HpSK•shikimate-3-phosphate•ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A•162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism.     

Crystal structures of shikimate dehydrogenase AroE from Thermus thermophilus HB8 and its cofactor and substrate complexes: insights into the enzymatic mechanism

Shikimate dehydrogenase (EC 1.1.1.25) catalyses the fourth step of the shikimate pathway which is required for the synthesis of the aromatic amino acids and other aromatic compounds in bacteria, microbial eukaryotes, and plants. The crystal structures of the shikimate dehydrogenase AroE from Thermus thermophilus HB8 in its ligand-free form, binary complexes with cofactor NADP+ or substrate shikimate, and the ternary complex with both NADP(H) and shikimate were determined by X-ray diffraction method at atomic resolutions. The crystals are nearly isomorphous with the asymmetric unit containing a dimer, each subunit of which has a bi-domain structure of compact alpha/beta sandwich folds. The two subunits of the enzyme display asymmetry in the crystals due to different relative orientations between the N- and C-terminal domains resulting in a slightly different closure of the interdomain clefts. NADP(H) is bound to the more closed form only. This closed conformation with apparent higher affinity to the cofactor is also observed in the unliganded crystal form, indicating that the NADP(H) binding to TtAroE may follow the selection mode where the cofactor binds to the subunit that happens to be in the closed conformation in solution. Crystal structures of the closed subunits with and without NADP(H) show no significant structural difference, suggesting that the cofactor binding to the closed subunit corresponds to the lock-and-key model in TtAroE. On the other hand, shikimate binds to both open and closed subunit conformers of both apo and NADP(H)-liganded holo enzyme forms. The ternary complex TtAroE:NADP(H):shikimate allows unambiguous visualization of the SDH permitting elucidation of the roles of conserved residues Lys64 and Asp100 in the hydride ion transfer between NADP(H) and shikimate.     

Conformational dynamics of the EGFR kinase domain reveals structural features involved in activation

The epidermal growth factor receptor (EGFR) has been the focus of intensive studies because of its importance in cancer research. Thus, a broader understanding of the molecular mechanism of activation of the EGFR kinase will have profound significance for the development of novel therapeutics. Numerous crystal structures of EGFR kinase, including the structure of the activating‐kinase dimer, have provided snapshots of the specific pathway. Herein, we performed unrestrained‐, as well as targeted‐molecular dynamics simulations based on these data, to gain further insight into the conformational changes responsible for activation. Comparison of the monomer‐ versus activating‐EGFR‐dimer simulations indicates that the dimerization is stabilizing structural elements associated with the activated state and predicts new salt‐bridge interactions involving activation‐loop residues that may also be associated with that state. Targeted molecular dynamics simulations of the inactive‐to‐active EGFR transition, as well as the reverse pathway, confirm the formation of conserved structural features of functional importance for the activity or stabilization of either conformation. Interestingly, simulations of the L834R mutant, which is associated with cancer, suggest that the structural basis of the activation induced by that mutation might be the ability of the mutated R834 residue to consecutively form salt bridges with neighboring acidic residues and cause destabilization of a hydrophobic cluster in the inactive state. Proteins 2009. © 2008 Wiley‐Liss, Inc.