Inhibitor-induced Conformational Changes Research Articles

Inhibition of Parkinson's disease-related LRRK2 by type I and type II kinase inhibitors: Activity and structures.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD) and a risk factor for the sporadic form. Increased kinase activity was shown in patients with both familial and sporadic PD, making LRRK2 kinase inhibitors a major focus of drug development efforts. Although much progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-electron microscopy structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type I inhibitor MLi-2 and the broad-spectrum type II inhibitor GZD-824. Our structures revealed an active-like LRRK2 kinase in the type I inhibitor complex, and an inactive DYG-out in the type II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structures provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.

Gaussian Accelerated Molecular Dynamics Simulations Investigation on the Mechanism of Angiotensin-Converting Enzyme (ACE) C-Domain Inhibition by Dipeptides.

Angiotensin-converting enzyme (ACE)-inhibitory peptides extracted from food proteins can lower blood pressure by inhibiting ACE activity. A recent study showed that the inhibitory activity of IY (Ile-Tyr, a dipeptide derived from soybean protein) against ACE was much higher than that of LL (Leu-Leu), although they had similar hydrophobic and predicted activity values. It was difficult to reveal the deep molecular mechanism underlying this phenomenon by traditional experimental methods. The Apo and two complex systems (i.e., ACE–LL and ACE–IY) were therefore subjected to 1 μs long Gaussian accelerated molecular dynamics (GaMD) simulations. The results showed that the binding of IY can cause obvious contraction of the active site of ACE, mainly manifested by a significant lateral shift of α13, α14, and α15. In addition, hinge 2 and hinge 3 were more stable in the ACE–IY system, while these phenomena were not present in the ACE–LL system. Moreover, the α10 of the IY-bound ACE kept an inward state during the simulation progress, which facilitated the ACE to remain closed. However, for the LL-bound ACE, the α10 switched between two outward states. To sum up, our study provides detailed insights into inhibitor-induced conformational changes in ACE that may help in the design of specific inhibitors targeting ACE for the treatment of hypertension.

All-Atomic Molecular Dynamic Studies of Human and Drosophila CDK8: Insights into Their Kinase Domains, the LXXLL Motifs, and Drug Binding Site.

Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, we analyzed the structures of human and Drosophila CDK8 proteins using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, we evaluated the structural differences between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, we have observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, our molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, we identified the inhibitor-induced conformational changes of hCDK8, and our results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.

Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

Inhibitor-Induced Conformational Changes in ASIC1A

Inhibitor-Induced Conformational Changes in ASIC1A

Structural Basis for KDM5A Histone Lysine Demethylase Inhibition by Diverse Compounds

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the invitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.

Application of Site-Specific Spin Labeling for NMR Detecting Inhibitor-Induced Conformational Change of HIV-1 Reverse Transcriptase.

Paramagnetism-assisted nuclear magnetic resonance (NMR) techniques can provide long-range structural information complemented with local information derived from chemical-shift perturbation and nuclear Overhauser effect data. Here, we address the application of paramagnetic relaxation enhancement (PRE) to detect inhibitor-induced conformational change of a drug target protein using human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) as a model protein. Using a site-specific spin-labeled HIV-1 RT mutant with selective (13) C labeling, conformation-dependent PREs were successfully observed reflecting the stabilization of an open conformation of this enzyme caused by inhibitor binding. This study demonstrates that the paramagnetism-assisted NMR approach offers an alternative strategy in protein-based drug screening to identify allosteric inhibitors of a target protein.

Molecular Dynamics Simulations of Sonic Hedgehog-Receptor and Inhibitor Complexes and Their Applications for Potential Anticancer Agent Discovery

The sonic hedgehog (Shh) signaling pathway is necessary for a variety of development and differentiation during embryogenesis as well as maintenance and renascence of diverse adult tissues. However, an abnormal activation of the signaling pathway is related to various cancers. In this pathway, the Shh signaling transduction is facilitated by binding of Shh to its receptor protein, Ptch. In this study, we modeled the 3D structure of functionally important key loop peptides of Ptch based on homologous proteins. Using this loop model, the molecular interactions between the structural components present in the pseudo-active site of Shh and key residues of Ptch was investigated in atomic level through molecular dynamics (MD) simulations. For the purpose of developing inhibitor candidates of the Shh signaling pathway, the Shh pseudo-active site of this interface region was selected as a target to block the direct binding between Shh and Ptch. Two different structure-based pharmacophore models were generated considering the key loop of Ptch and known inhibitor-induced conformational changes of the Shh through MD simulations. Finally two hit compounds were retrieved through a series of virtual screening combined with molecular docking simulations and we propose two hit compounds as potential inhibitory lead candidates to block the Shh signaling pathway based on their strong interactions to receptor or inhibitor induced conformations of the Shh.

On the Mechanism of CFTR Inhibition by a Thiazolidinone Derivative

We employed inside-out patch-clamp technique to investigate the mechanism of CFTR inhibition by a thiazolidinone derivative, CFTRinh-172. We found that application of CFTRinh-172 results in an increase of the mean closed time, and a decrease of the mean open time of WT-CFTR channels. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. We have shown that though CFTRinh-172 can bind to both open and closed state of the channel, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance following binding of the inhibitor to the channel. Our data also indicate that stabilization of the open state leads to more potent inhibition: K1/2 is reduced to nanomolar range for locked-open CFTR channels. Interestingly, we found that although there is >95% homology between pig and human proteins, pig CFTR is significantly less sensitive to CFTRinh-172 (K1/2 = 8.05 ± 3.38 μM for pig CFTR vs K1/2 = 1.24 ± 0.14 μM for human CFTR). This result is especially puzzling since the open time for pig CFTR is ∼5 times longer than that of human CFTR. Like its human counterpart, pig CFTR, locked open with PPi, becomes more sensitive to CFTRinh-172 (K1/2 = 2.21 ± 0.41 μM). However, compared to human CFTR locked open with PPi, pig channels are >100 fold less sensitive to the inhibitor. We also found that dog CFTR behaves very similarly to pig CFTR, including a longer open time and a lower sensitivity to CFTRinh-172. Sequence analysis and chimera approach allowed us to reveal the region between the R- and NBD2- domains as largely responsible for this difference in sensitivity. Experiments further identifying responsible amino acid residue(s) are under way.

On the mechanism of CFTR inhibition by a thiazolidinone derivative

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172–dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels “inactivate” despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

Dynamics of Glyphosate-Induced Conformational Changes of Mycobacterium tuberculosis 5-Enolpyruvylshikimate-3-phosphate Synthase (EC 2.5.1.19) Determined by Hydrogen−Deuterium Exchange and Electrospray Mass Spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis ( Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Rational Design of Non-Nucleoside, Potent, and Orally Bioavailable Adenosine Deaminase Inhibitors: Predicting Enzyme Conformational Change and Metabolism

From metabolic considerations and prediction of an inhibitor-induced conformational change, novel adenosine deaminase (ADA) inhibitors with improved activities and oral bioavailability have been developed on the basis of our originally designed non-nucleoside ADA inhibitors. They demonstrated in vivo efficacy in models of inflammation and lymphoma. Furthermore, X-ray crystal structure analysis has revealed a novel induced fit to ADA.

Slow Tight Binding Inhibition of Proteinase K by a Proteinaceous Inhibitor: CONFORMATIONAL ALTERATIONS RESPONSIBLE FOR CONFERRING IRREVERSIBILITY TO THE ENZYME-INHIBITOR COMPLEX

The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.

Evidence for Distinct Sodium-, Dopamine-, and Cocaine-dependent Conformational Changes in Transmembrane Segments 7 and 8 of the Dopamine Transporter

Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.

Probing Inhibitor-Induced Conformational Changes along the Interface between Tissue Factor and Factor VIIa

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF--FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

Crystal structure of carboxypeptidase A complexed with D-cysteine at 1.75 A - inhibitor-induced conformational changes.

D-Cysteine differs from the antiarthritis drug D-penicillamine by only two methyl groups on the beta-carbon yet inhibits carboxypeptidase A (CPD) by a distinct mechanism: D-cysteine binds tightly to the active site zinc, while D-penicillamine catalyzes metal removal. To investigate the structural basis for this difference, we solved the crystal structure of carboxypeptidase A complexed with D-cysteine (D-Cys) at 1.75-A resolution. D-Cys binds the active site zinc with a sulfur ligand and forms additional interactions with surrounding side chains of the enzyme. The structure explains the difference in potency between D-Cys and L-Cys and provides insight into the mechanism of D-penicillamine inhibition. D-Cys binding induces a concerted motion of the side chains around the zinc ion, similar to that found in other carboxypeptidase-inhibitor crystal structures and along a limited path. Analysis of concerted motions of CPD and CPD-inhibitor crystal structures reveals a clustering of these structures into distinct groups. Using the restricted conformational flexibility of a drug target in this type of analysis could greatly enhance efficiency in drug design.

Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.

AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonate) and DNDS (4,4'-dinitrostilbene-2,2'-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes upon noncovalent binding of DNDS. Since conformational changes induced by stilbene disulfonate binding cause anion transport inhibition, identification of the DNDS binding regions may localize the substrate binding region of the protein. Cysteine residues were introduced into 27 sites in the extracellular loop regions of an otherwise cysteineless form of AE1, called AE1C(-). The ability to label these residues with biotin maleimide [3-(N-maleimidylpropionyl)biocytin] was then measured in the absence and presence of DNDS. DNDS reduced the ability to label residues in the regions around G565, S643-M663, and S731-S742. We interpret these regions either as (i) part of the DNDS binding site or (ii) distal to the binding site but undergoing a conformational change that sequesters the region from accessibility to biotin maleimide. DNDS alters the conformation of residues outside the plane of the bilayer since the S643-M663 region was previously shown to be extramembranous. Upon binding DNDS, AE1 undergoes conformational changes that can be detected in extracellular loops at least 20 residues away from the hydrophobic core of the lipid bilayer. We conclude that the TM7-10 region of AE1 is central to the stilbene disulfonate and substrate binding region of AE1.

Crystal structure of the stromelysin catalytic domain at 2.0 Å resolution: inhibitor-induced conformational changes

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83–255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three α-helices and one twisted, five-strand β-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1′ specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1′ cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1′ cavity, while in the complex, the P1′ group of the inhibitor displaces the Tyr223 in order to fit into the S1′ cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1′ cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251–255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.

Substrate- and Inhibitor-induced Conformational Changes in the Yeast V-ATPase Provide Evidence for Communication between the Catalytic and Proton-translocating Sectors

The vacuolar-type H(+)-ATPases (V-ATPases) are composed of two distinct sectors, a catalytic complex (V(1)) involved in ATP hydrolysis and a membrane-associated complex (V(0)) mediating proton translocation across a lipid bilayer. To date, little is known about the mechanism by which these two functions are coupled. We sought to examine the impact of nucleotide and cation binding on the structure of the core components of the catalytic complex and to determine whether conformational changes within the catalytic complex impact subunits of the membrane-associated complex. Nucleotide- and cation- induced changes in the catalytic core of the V-ATPase were investigated by monitoring changes in the rate and pattern of tryptic digests. ATP.Mg-induced changes were detected in both the catalytic (Vma1p or 69 kDa) and the regulatory subunits (Vma2p or 60 kDa) of the V(1) sector. ATP alone increased the rate of trypsinization of the regulatory subunit, but did not have any effect on Vma1p. Surprisingly, ATP also had an impact on the 95-kDa subunit, a component of the V(0) sector of the V-ATPase. Although the presence of divalent cations had no impact on the V(1) sector, the rate of trypsinization of the 95-kDa subunit was greatly enhanced. The effect of divalent cations on the structure of the 95-kDa subunit was abrogated when trypsinization was performed in the absence of the catalytic sector. Addition of bafilomycin A(1), a V-ATPase inhibitor that putatively binds to the 95-kDa subunit, increased the rate of trypsinization of the catalytic subunit. These data suggest that structural alterations within the V(1) sector result in alterations within the V(0) sector and vice versa. Clearly, a structural link must exist to couple the two sectors. The 95-kDa subunit is ideally suited to fulfill this role. Hydropathy analysis suggests a bipartite structure, with the NH(2)-terminal portion predicted to lie in an aqueous environment and the C-terminal portion predicted to contain 6 transmembrane segments. Tryptic digests of sealed vacuolar vesicles and immunofluorescence studies revealed that the large hydrophilic NH(2)-terminal domain of the 95-kDa subunit is localized toward the cytosol. This region therefore is ideally positioned to interact with components of the V(1) complex, potentially functioning as the elusive link between the two sectors of the V-ATPase.

Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS).

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional topology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon binding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the structural and mechanistic similarities of MurA and EPSPS, a similar conformational change is likely to occur in EPSPS to generate a catalytically competent active site. However, there is currently little experimental evidence available to support the occurrence of such a conformational change in EPSPS. Using limited tryptic digestion of MurA,(1) it could be shown that formation of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed conformation was achieved by addition of either an excess of both substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomycin. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry demonstrates that the protection of the protein in either case is caused by (1) a specific shielding of regions thereby becoming less accessible as a result of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the peptide backbone in the binary and ternary complexes. The establishment of methods to describe the effects of tryptic digestion on MurA under various conditions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of proteolytic degradation. When unliganded EPSPS was treated with trypsin, three of the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a conformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained using a conformational sensitive monoclonal antibody against EPSPS which showed a 20-fold reduced affinity toward the protein complexed with S3P and glyphosate as compared to the unliganded enzyme.