Dennis W. Wolan

Domain Swapping in Inducible Nitric-oxide Synthase ELECTRON TRANSFER OCCURS BETWEEN FLAVIN AND HEME GROUPS LOCATED ON ADJACENT SUBUNITS IN THE DIMER

Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and l-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis froml-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 7309–7312). Here, we characterize a pair of heterodimers that contain an l-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented l-Arg binding to one oxygenase domain in each heterodimer but did not affect thel-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-l-Arg interaction confirmed that E371A only prevents l-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.

Small-Molecule Activators of a Proenzyme

Small-Molecule Protease Activator Human proteases regulate numerous biological processes. Most are stored as inactive proenzymes that are activated either by upstream processes or by self-proteolysis. Wolan et al. (p. 853) have identified a small molecule that activates the apoptotic procaspases-3 and -6. These caspases are homodimers, and the compound probably competitively inhibits one active site, but stabilizes an on-state conformation that promotes self-cleavage by the unoccupied site. It may thus be possible to find other small-molecule activators for other proenzymes that should facilitate functional and mechanistic studies. Small molecules that promote a procaspase conformation susceptible to activation by proteolysis have been identified. Virtually all of the 560 human proteases are stored as inactive proenyzmes and are strictly regulated. We report the identification and characterization of the first small molecules that directly activate proenzymes, the apoptotic procaspases-3 and -6. It is surprising that these compounds induce autoproteolytic activation by stabilizing a conformation that is both more active and more susceptible to intermolecular proteolysis. These procaspase activators bypass the normal upstream proapoptotic signaling cascades and induce rapid apoptosis in a variety of cell lines. Systematic biochemical and biophysical analyses identified a cluster of mutations in procaspase-3 that resist small-molecule activation both in vitro and in cells. Compounds that induce gain of function are rare, and the activators reported here will enable direct control of the executioner caspases in apoptosis and in cellular differentiation. More generally, these studies presage the discovery of other proenzyme activators to explore fundamental processes of proenzyme activation and their fate-determining roles in biology.

Proteome-wide covalent ligand discovery in native biological systems

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered ‘undruggable’. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand–protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Turning a protein kinase on or off from a single allosteric site via disulfide trapping.

There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)—the PDK1-interacting-fragment (PIF) pocket—is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation. We also discovered a variety of small molecule fragment disulfides (< 300 Da) that could either activate or inhibit PDK1 by conjugation to the PIF pocket, thus displaying greater functional diversity than is displayed by PIFtides conjugated to the same sites. Biochemical data and three crystal structures provided insight into the mechanism of action of the best fragment activators and inhibitors. These studies show that disulfide trapping is useful for characterizing allosteric sites on kinases and that a single allosteric site on a protein kinase can be exploited for both activation and inhibition by small molecules.

Crystal structure of the murine NK cell–activating receptor NKG2D at 1.95 Å

NKG2D, a homodimeric lectin-like receptor, is a unique stimulatory molecule that is found on natural killer cells, T cells and activated macrophages. The natural ligands for murine NKG2D are distant major histocompatibility complex homologs, retinoic acid early transcript (Rae1) and H-60 minor histocompatibility antigen. The crystal structure of the extracellular region of murine NKG2D reveals close homology with other C-type lectin receptors such as CD94, Ly49A, rat MBP-A and CD69. However, the precise mode of dimeric assembly varies among these natural killer receptors, as well as their surface topography and electrostatic properties. The NKG2D structure provides the first structural insights into the role and ligand specificity of this stimulatory receptor in the innate and adaptive immune system.

Antifungal Imidazoles Block Assembly of Inducible NO Synthase into an Active Dimer

Cytokine-inducible nitric oxide synthase (iNOS) is a homodimeric enzyme that generates nitric oxide (NO) andl-citrulline from l-arginine (l-Arg) and O2. The N-terminal oxygenase domain (amino acids 1–498; iNOSox) in each subunit binds heme,l-Arg, and tetrahydrobiopterin (H4B), is the site of NO synthesis, and is responsible for the dimeric interaction, which must occur to synthesize NO. In both cells and purified systems, iNOS dimer assembly is promoted by H4B, l-Arg, and l-Arg analogs. We examined the ability of imidazole andN-substituted imidazoles to promote or inhibit dimerization of heme-containing iNOSox monomers, or to affect iNOS dimerization in cells. Imidazole, 1-phenylimidazole, clotrimazole, and miconazole all bound to the iNOSox monomer heme iron. Imidazole and 1-phenylimidazole promoted iNOSox dimerization, whereas clotrimazole (30 μm) and miconazole (15 μm) did not, and instead inhibited dimerization normally promoted byl-Arg and H4B. Clotrimazole also bound to iNOSox dimers in the absence of l-Arg and H4B and caused their dissociation. When added to cells expressing iNOS, clotrimazole (50 μm) had no effect on iNOS protein expression but almost completely inhibited its dimerization and consequent NO synthesis over an 8-h culture period, without affecting calmodulin interaction with iNOS. Thus, imidazoles can promote or inhibit dimerization of iNOS both in vitro and in cells, depending on their structure. Bulky imidazoles like clotrimazole block NO synthesis by inhibiting assembly of the iNOS dimer, revealing a new means to control cellular NO synthesis.

Self-Assembling Small Molecules Form Nanofibrils That Bind Procaspase-3 to Promote Activation

Modulating enzyme function with small-molecule activators, as opposed to inhibitors, offers new opportunities for drug discovery and allosteric regulation. We previously identified a compound, called 1541, from a high-throughput screen (HTS) that stimulates activation of a proenzyme, procaspase-3, to generate mature caspase-3. Here we further investigate the mechanism of activation and report the surprising finding that 1541 self-assembles into nanofibrils exceeding 1 μm in length. These particles are an unanticipated outcome from an HTS that have properties distinct from standard globular protein aggregators. Moreover, 1541 nanofibrils function as a unique biocatalytic material that activates procaspase-3 via induced proximity. These studies demonstrate a novel approach for proenzyme activation through binding to fibrils, which may mimic how procaspases are naturally processed on protein scaffolds.

Inducible nitric oxide synthase: role of the N‐terminal β‐hairpin hook and pterin‐binding segment in dimerization and tetrahydrobiopterin interaction

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1–498) is a dimer that binds heme, L‐arginine and tetrahydrobiopterin (H4B) and is the site for nitric oxide synthesis. We examined an N‐terminal segment that contains a β‐hairpin hook, a zinc ligation center and part of the H4B‐binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N‐terminal β‐hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side‐chain hydrogen bonds and packing interactions with the iNOSox C‐terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H4B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C‐terminus and cause similar defects that were reversible with high concentrations of H4B. Heterodimers comprised of a hook‐defective iNOSox mutant subunit and a full‐length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N‐terminal hooks exchange between subunits in solution to stabilize the dimer.

Mutational Analysis of the Tetrahydrobiopterin-binding Site in Inducible Nitric-oxide Synthase

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.

Acetone‐Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling

Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAseu2005S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags.