Distinct dimer interaction and regulation in nitric-oxide synthase types I,II, and III

Homodimer formation activates all nitric-oxide synthases (NOSs). It involves the interaction between two oxygenase domains (NOSoxy) that each bind heme and (6R)-tetrahydrobiopterin (H4B) and catalyze NO synthesis from L-Arg. Here we compared three NOSoxy isozymes regarding dimer strength, interface composition, and the ability of L-Arg and H4B to stabilize the dimer, promote its formation, and protect it from proteolysis. Urea dissociation studies indicated that the relative dimer strengths were NOSIIIoxy > NOSIoxy > NOSIIoxy (endothelial NOSoxy (eNOSoxy) > neuronal NOSOXY (nNOSoxy) > inducible NOSoxy (iNOSoxy)). Dimer strengths of the full-length NOSs had the same rank order as judged by their urea-induced loss of NO synthesis activity. NOSoxy dimers containing L-Arg plus H4B exhibited the greatest resistance to urea-induced dissociation followed by those containing either molecule and then by those containing neither. Analysis of crystallographic structures of eNOSoxy and iNOSoxy dimers showed more intersubunit contacts and buried surface area in the dimer interface of eNOSoxy than iNOSoxy, thus revealing a potential basis for their different stabilities. L-Arg plus H4B promoted dimerization of urea-generated iNOSoxy and nNOSoxy monomers, which otherwise was minimal in their absence, and also protected both dimers against trypsin proteolysis. In these respects, L-Arg alone was more effective than H4B alone for nNOSoxy, whereas for iNOSoxy the converse was true. The eNOSoxy dimer was insensitive to proteolysis under all conditions. Our results indicate that the three NOS isozymes, despite their general structural similarity, differ markedly in their strengths, interfaces, and in how L-Arg and H4B influence their formation and stability. These distinguishing features may provide a basis for selective control and likely help to regulate each NOS in its particular biologic milieu.     

Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms

The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) cofactor and l-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition of l-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.     

The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from l-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s(-1)) > iNOSoxy (11 s(-1)) > eNOSoxy (6 s(-1)), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical.     

Implications for isoform-selective inhibitor design derived from the binding mode of bulky isothioureas to the heme domain of endothelial nitric-oxide synthase

Nitric oxide produced by nitric-oxide synthase (NOS) is not only involved in a wide range of physiological functions but also in a variety of pathological conditions. Isoform-selective NOS inhibitors are highly desirable to regulate the NO production of one isoform beneficial to normal physiological functions from the uncontrolled NO production of another isoform that accompanies certain pathological states. Crystal structures of the heme domain of the three NOS isoforms have revealed a very high degree of similarity in the immediate vicinity of the heme active site illustrating the challenge of isoform-selective inhibitor design. Isothioureas are potent NOS inhibitors, and the structures of the endothelial NOS heme domain complexed with isothioureas bearing small S-alkyl substituents have been determined (Li, H., Raman, C.S., Martásek, P., Král, V., Masters, B.S.S., and Poulos, T.L. (2000) J. Inorg. Biochem. 81, 133--139). In the present communication, the binding mode of larger bisisothioureas complexed to the endothelial NOS heme domain has been determined. These structures afford a structural rationale for the known inhibitory activities. In addition, these structures provide clues on how to exploit the longer inhibitor substituents that extend out of the active site pocket for isoform-selective inhibitor design.     

Structural insights into the binding of MMP9 inhibitors

Matrix Metalloproteinase are family of enzymes responsible for degradation of extracellular matrix. MMP9 (gelatinase B) is one of the common matrix metalloproteinase that is associated with tissue destruction in a number of disease states such as rheumatoid arthiritis, fibrotic lung disease, dilated cardiomyopathy, as well as cancer invasion and metastasis. Recent study demonstrates that increased expression of MMP9 results in augmentation of myopathy with increased inflammation and fibernecrosis. Previous studies do not provide any conclusive information related to structural specificity of MMP9 inhibitors towards its active site, but with the availability of experimental structures it is now possible to study the structural specificity of MMP9 inhibitors. In light of availability of this information, we have applied docking and molecular dynamics approach to study the binding of inhibitors to the active site of MMP9. Three categories of inhibitor consisting of sulfonamide hydroxamate, thioester, and carboxylic moieties as zinc binding groups (ZBG) were chosen in the present study. Our docking results demonstrate that thioester based zinc binding group gives favourable docking scores as compared to other two groups. Molecular Dynamics simulations further reveal that tight binding conformation for thioester group has high specificity for MMP9 active site. Our study provides valuable insights on inhibitor specificity of MMP9 which provides valuable hints for future design of potent inhibitors and drugs.     

Structure of testis ACE glycosylation mutants and evidence for conserved domain movement

Human angiotensin-converting enzyme is an important drug target for which little structural information has been available until recent years. The slow progress in obtaining a crystal structure was due to the problem of surface glycosylation, a difficulty that has thus far been overcome by the use of a glucosidase-1 inhibitor in the tissue culture medium. However, the prohibitive cost of these inhibitors and incomplete glucosidase inhibition makes alternative routes to minimizing the N-glycan heterogeneity desirable. Here, glycosylation in the testis isoform (tACE) has been reduced by Asn-Gln point mutations at N-glycosylation sites, and the crystal structures of mutants having two and four intact sites have been solved to 2.0 A and 2.8 A, respectively. Both mutants show close structural identity with the wild-type. A hinge mechanism is proposed for substrate entry into the active cleft, based on homology to human ACE2 at the levels of sequence and flexibility. This is supported by normal-mode analysis that reveals intrinsic flexibility about the active site of tACE. Subdomain II, containing bound chloride and zinc ions, is found to have greater stability than subdomain I in the structures of three ACE homologues. Crystallizable glycosylation mutants open up new possibilities for cocrystallization studies to aid the design of novel ACE inhibitors.     

Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: A basis for the design of specific inhibitors

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives N(omega)-methyl-L-arginine (MMA) and N(omega),N(omega)-dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.     

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1′ pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.     

Complexes of tRNA and maturation enzymes: shaping up for translation

Several significant structures of transfer ribonucleic acid (tRNA) maturation enzymes complexed with precursor tRNA or fragments thereof have been published recently, providing detailed knowledge of enzyme-tRNA recognition and catalytic strategies. In addition to reinforcing the general principles of RNA-protein interaction, the new structures highlight both the features of composite RNA recognition by multiple enzyme subunits and the pronounced RNA structural flexibility in or near the active site in all cases. These structural principles provide plausible explanations for the exquisite specificity and catalytic power of these enzymes and, in the case of evolutionary adaptation, for the ability of some enzymes to develop novel specificities.     

The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

Dissecting the catalytic mechanism of a plant beta-D-glucan glucohydrolase through structural biology using inhibitors and substrate analogues

Higher plant, family GH3 beta-D-glucan glucohydrolases exhibit exo-hydrolytic and retaining (e-->e) mechanisms of action and catalyze the removal of single glucosyl residues from the non-reducing termini of beta-D-linked glucosidic substrates, with retention of anomeric configuration. The broad specificity beta-D-glucan glucohydrolases are likely to play roles in cell wall re-modelling, turn-over of cell wall components and possibly in plant defence reactions against pathogens. Crystal structures of the barley beta-D-glucan glucohydrolase, obtained from both native enzyme and from the enzyme in complex with a substrate analogues and mechanism-based inhibitors, have enabled the basis of substrate specificity, the mechanism of catalysis, and the role of domain movements during the catalytic cycle to be defined in precise molecular terms. The active site of the enzyme forms a shallow 'pocket' that is located at the interface of two domains of the enzyme and accommodates two glucosyl residues. The propensity of the enzyme to hydrolyze a broad range of substrates with (1-->2)-, (1-->3)-, (1-->4)- and (1-->6)-beta-D-glucosidic linkages is explained from crystal structures of the enzyme in complex with non-hydrolysable S-glycoside substrate analogues, and from molecular modelling. During binding of gluco-oligosaccharides, the glucosyl residue at subsite -1 is locked in a highly constrained position, but the glucosyl residue at the +1 subsite is free to adjust its position between two tryptophan residues positioned at the entry of the active site pocket. The flexibility at subsite +1 and the projection of the remainder of the substrate away from the pocket provide a structural rationale for the capacity of the enzyme to accommodate and hydrolyze glucosides with different linkage positions and hence different overall conformations. While mechanism-based inhibitors with micromolar Ki constants bind in the active site of the enzyme and form esters with the catalytic nucleophile, transition-state mimics bind with their 'glucose' moieties distorted into the 4E conformation, which is critical for the nanomolar binding of these inhibitors to the enzyme. The glucose product of the reaction, which is released from the non-reducing termini of substrates, remains bound to the beta-D-glucan glucohydrolase in the -1 subsite of the active site, until a new substrate molecule approaches the enzyme. If dissociation of the glucose from the enzyme active site could be synchronized throughout the crystal, time-resolved Laue X-ray crystallography could be used to follow the conformational changes that occur as the glucose product diffuses away and the incoming substrate is bound by the enzyme.     

Structures of the neuronal and endothelial nitric oxide synthase heme domain with D-nitroarginine-containing dipeptide inhibitors bound

In a continuing effort to unravel the structural basis for isoform-selective inhibition of nitric oxide synthase (NOS) by various inhibitors, we have determined the crystal structures of the nNOS and eNOS heme domain bound with two D-nitroarginine-containing dipeptide inhibitors, D-Lys-D-Arg(NO)2-NH(2) and D-Phe-D-Arg(NO)2-NH(2). These two dipeptide inhibitors exhibit similar binding modes in the two constitutive NOS isozymes, which is consistent with the similar binding affinities for the two isoforms as determined by K(i) measurements. The D-nitroarginine-containing dipeptide inhibitors are not distinguished by the amino acid difference between nNOS and eNOS (Asp 597 and Asn 368, respectively) which is key in controlling isoform selection for nNOS over eNOS observed for the L-nitroarginine-containing dipeptide inhibitors reported previously [Flinspach, M., et al. (2004) Nat. Struct. Mol. Biol. 11, 54-59]. The lack of a free alpha-amino group on the D-nitroarginine moiety makes the dipeptide inhibitor steer away from the amino acid binding pocket near the active site. This allows the inhibitor to extend into the solvent-accessible channel farther away from the active site, which enables the inhibitors to explore new isoform-specific enzyme-inhibitor interactions. This might be the structural basis for why these D-nitroarginine-containing inhibitors are selective for nNOS (or eNOS) over iNOS.     

Structural organization in the serine proteases. I. Macromolecular specificity in limited proteolysis

We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered about the serine proteases. The emphasis of these approaches is to compare and contrast macromolecules at the separate levels of secondary, tertiary, and quaternary structure. Our assumption is that in functionally related molecules, regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of specificity. Based on this assumption, the independent observations from studies of these enzymes in solution and in biological systems are combined with the structural observations from X-ray crystallographic analysis. A goal of the present research effort is to probe the biomolecular architecture of the serine proteases to evaluate the role of the three-dimensional structure beyond that of the active site in determining recognition and reactivity determinants for these enzymes, and to determine those principles that might be applied successfully to other enzyme systems as well. Of particular note has been our observation of a macromolecular recognition surface which occurs as a topographical feature outside of the active site and under evolutionary control to produce specificity towards macromolecular substrates and inhibitors. In addition we have established the important role of conformational changes that occur beyond the active site of an enzyme and differentiate between natural and synthetic inhibitor-enzyme interactions. This suggests that the specificity and reactivity determinants of a macromolecule are derived from its architecture and structural organization.     

The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase

The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy). We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2). This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.     

Identification of two residues essential for the stringent substrate specificity and active site stability of the prokaryotic l-arginine:glycine amidinotransferase CyrA

A novel prokaryotic l-arginine:glycine amidinotransferase (CyrA; EC2.1.4.1) is involved in the biosynthesis of the polyketide-derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J277, 3844-3860]. In order to elucidate the structure-function-stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human l-arginine:glycine amidinotransferase (h-AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h-AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand-induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand-induced structural changes that involve Phe245. Stability parameters (T(opt) and T(max) ) of the CyrA variants differed from those of wild-type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in T(opt) are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme.     

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S1 pocket of SARS-CoV Mpro

The 3C-like main proteinase of the severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV M(pro), is widely considered to be a major drug target for the development of anti-SARS treatment. Based on the chemical structure of a lead compound from a previous screening, we have designed and synthesized a number of non-peptidyl inhibitors, some of which have shown significantly improved inhibitory activity against SARS-CoV M(pro) with IC(50) values of approximately 60 nM. In the absence of SARS-CoV M(pro) crystal structures in complex with these synthetic inhibitors, molecular docking tools have been employed to study possible interactions between these inhibitors and SARS-CoV M(pro). The docking results suggest two major modes for the initial binding of these inhibitors to the active site of SARS-CoV M(pro). They also establish a structural basis for the 'core design' of these inhibitors by showing that the 3-chloropyridine functions common to all of the present inhibitors tend to cluster in the S1 specificity pocket. In addition, intrinsic flexibility in the S4 pocket allows for the accommodation of bulky groups such as benzene rings, suggesting that this structural plasticity can be further exploited for optimizing inhibitor-enzyme interactions that should promote a tighter binding mode. Most importantly, our results provide the structural basis for rational design of wide-spectrum antiviral drugs targeting the chymotrypsin-like cysteine proteinases from coronaviruses and picornaviruses.     

Conformational flexibility in crystal structures of human 11beta-hydroxysteroid dehydrogenase type I provide insights into glucocorticoid interconversion and enzyme regulation

Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) is an ER-localized membrane protein that catalyzes the interconversion of cortisone and cortisol. In adipose tissue, excessive cortisol production through 11beta-HSD1 activity has been implicated in the pathogenesis of type II diabetes and obesity. We report here biophysical, kinetic, mutagenesis, and structural data on two ternary complexes of 11beta-HSD1. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11beta-HSD1 C termini converge to form an as yet uncharacterized tetramerization motif. A C-terminal Pro-Cys motif is localized at the center of the tetramer and forms reversible enzyme disulfides that alter enzyme activity. Conformational flexibility at the tetramerization interface is coupled to structural changes at the enzyme active site suggesting how the central Pro-Cys motif may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding 11beta-HSD1 activities and will ultimately facilitate the development of specific inhibitors.