High-Resolution Structure and Inhibition of the Schizophrenia-Linked Pseudokinase ULK4.

The ULK (UNC51-like) enzymes are a family of mammalian kinases that have critical roles in autophagy and development. While ULK1, ULK2, and ULK3 have been characterized, very little is known about ULK4. However, recently, deletions in ULK4 have been genetically linked to increased susceptibility to developing schizophrenia, a devastating neuropsychiatric disease with high heritability but few genes identified. Interestingly, ULK4 is a pseudokinase with some unusual mutations in the kinase catalytic motifs. Here, we report the first structure of the human ULK4 kinase at high resolution and show that although ULK4 has no apparent phosphotransfer activity, it can strongly bind ATP. We find an unusual mechanism for binding ATP in a Mg2+-independent manner, including a rare hydrophobic bridge in the active site. In addition, we develop two assays for ATP binding to ULK4, perform a virtual and experimental screen to identify small-molecule binders of ULK4, and identify several novel scaffolds that bind ULK4 and can lead the way to more selective small molecules that may help shed light on the function of this enigmatic protein.

Structure-Activity Relationships and Biological Evaluation of 7-Substituted Harmine Analogs for Human ß-Cell Proliferation.

Recently, we have shown that harmine induces ß-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. We explore structure-activity relationships of the 7-position of harmine for both DYRK1A kinase inhibition and ß-cell proliferation based on our related previous structure-activity relationship studies of harmine in the context of diabetes and ß-cell specific targeting strategies. 33 harmine analogs of the 7-position substituent were synthesized and evaluated for biological activity. Two novel inhibitors were identified which showed DYRK1A inhibition and human ß-cell proliferation capability. The DYRK1A inhibitor, compound 1-2b, induced ß-cell proliferation half that of harmine at three times higher concentration. From these studies we can draw the inference that 7-position modification is limited for further harmine optimization focused on ß-cell proliferation and cell-specific targeting approach for diabetes therapeutics.

A conserved tryptophan (W91) at the barrel-lid junction modulates the packing and stability of Kunitz (STI) family of inhibitors.

ß-trefoil fold, consisting of a six stranded ß-barrel capped at one end by a lid comprising of another six ß-strands, is one of the most important folds among proteins. Important classes of proteins like Interleukins (ILs), Fibroblast Growth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Their core is packed by hydrophobic residues contributed by the 6 stranded ß-barrel and three ß-hairpins that make essential contacts with each other and keep the protein in 'topologically minimal frustrated state'. A complete database analysis of the core residues of the ß-trefoil fold proteins presented here identified a conserved tryptophan (W91) residue in the Kunitz (STI) family of inhibitors that projects from the lid and interacts with the bottom layer residues of the barrel. This kind of interactions is unique in Kunitz (STI) family because no other families of ß-trefoil fold have such a shear sized residue at the barrel lid junction; suggesting its possible importance in packing and stability. We took WCI as a representative of this family and prepared four cavity creating mutants W91F-WCI, W91M-WCI, W91I-WCI & W91A-WCI. CD experiments show that the secondary structure of the mutants remains indistinguishable with the wild type. Crystal structures of the mutants W91F-WCI, W91M-WCI & W91A-WCI also show the same feature. However, slight readjustments of the side chains around the site of mutation have been observed so as to minimize the cavity created due to mutation. Comparative stability of these mutants, estimated using heat denaturation CD spectroscopy, indicates that stability of the mutants inversely correlates with the size of the cavity inside the core. Interestingly, although we mutated at the core, mutants show varying susceptibility against tryptic digestion that grossly follow their instability determined by CD. Our findings suggest that the W91 residue plays an important role in determining the stability and packing of the core of WCI.

Characterization, Structure, and Inhibition of the Human Succinyl-CoA:glutarate-CoA Transferase, a Putative Genetic Modifier of Glutaric Aciduria Type 1.

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.

Characterization, structure and inhibition of the human succinyl-CoA:glutarate-CoA transferase, a genetic modifier of glutaric aciduria type 1.

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or non-toxic metabolites. Here, we report a novel target, SUGCT, which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA. We report the structure of SUGCT, the first eukaryotic structure of a type III CoA transferase, develop a high-throughput enzyme assay and a cell-based assay, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme validating the screening approach. These results may form the basis for future development of new pharmacological intervention to treat GA1.

The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.

Psu is a capsid decoration protein of bacteriophage P4 and acts as an antiterminator of Rho-dependent transcription termination in bacteria. So far, no structures have been reported for the Psu protein or its homologues. Here, we report the first structure of Psu solved by the Hg(2+) single wavelength anomalous dispersion method, which reveals that Psu exists as a knotted homodimer and is first of its kind in nature. Each monomer of Psu attains a novel fold around a tight coiled-coil motif. CD spectroscopy and the structure of an engineered disulfide-bridged Psu derivative reveal that the protein folds reversibly and reassembles by itself into the knotted dimeric conformation without the requirement of any chaperone. This structure would help to explain the functional properties of the protein and can be used as a template to design a minimal peptide fragment that can be used as a drug against Rho-dependent transcription termination in bacteria.

Role of remote scaffolding residues in the inhibitory loop pre-organization, flexibility, rigidification and enzyme inhibition of serine protease inhibitors.

Canonical serine protease inhibitors interact with cognate enzymes through the P3-P2' region of the inhibitory loop while its scaffold hardly makes any contact. Neighboring scaffolding residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETI(L)-WCI(S), having a loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which cannot act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETI(L)-WCI(S)), where the barrier is reintroduced on ETI(L)-WCI(S), shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETI(L)-WCI(S)) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing the primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.

Structural insights into RNA cleavage by a novel family of bacterial RNases.

Processing of RNA is a key regulatory mechanism for all living systems. We recently discovered a novel family of endoribonucleases that is conserved across all bacteria. Here, using crystallography, cryo-EM microscopy, biochemical, biophysical, and mass spectrometry techniques, we are able to shed light on a novel RNA cleavage mechanism in bacteria. We show that YicC, the prototypical member of this family, forms a hexameric channel that closes down on a 26-mer RNA substrate, and find that it cleaves across an RNA hairpin to generate several short fragments.

Cloning, purification, crystallization and preliminary X-ray analysis of two low-molecular-weight protein tyrosine phosphatases from Vibrio cholerae.

Low-molecular-weight protein tyrosine phosphatases (LMWPTPs) are small cytoplasmic enzymes of molecular weight ∼18 kDa that belong to the large family of protein tyrosine phosphatases (PTPs). Despite their wide distribution in both prokaryotes and eukaryotes, their exact biological role in bacterial systems is not yet clear. Two low-molecular-weight protein tyrosine phosphatases (VcLMWPTP-1 and VcLMWPTP-2) from the Gram-negative bacterium Vibrio cholerae have been cloned, overexpressed, purified by Ni(2+)-NTA affinity chromatography followed by gel filtration and used for crystallization. Crystals of VcLMWPTP-1 were grown in the presence of ammonium sulfate and glycerol and diffracted to a resolution of 1.6 Å. VcLMWPTP-2 crystals were grown in PEG 4000 and diffracted to a resolution of 2.7 Å. Analysis of the diffraction data showed that the VcLMWPTP-1 crystals had symmetry consistent with space group P3(1) and that the VcLMWPTP-2 crystals had the symmetry of space group C2. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient for the VcLMWPTP-1 crystals was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%. The corresponding values for the VcLMWPTP-2 crystals, assuming the presence of two molecules in the asymmetric unit, were 2.77 Å(3) Da(-1) and 55.62%, respectively.

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1.

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.

Overexpression, purification, crystallization and preliminary X-ray analysis of CheY4 from Vibrio cholerae O395.

Chemotaxis and motility greatly influence the infectivity of Vibrio cholerae, although the role of chemotaxis genes in V. cholerae pathogenesis is poorly understood. In contrast to the single copy of CheY found in Escherichia coli and Salmonella typhimurium, four CheYs (CheY1-CheY4) are present in V. cholerae. While insertional disruption of the cheY4 gene results in decreased motility, insertional duplication of this gene increases motility and causes enhanced expression of the two major virulence genes. Additionally, cheY3/cheY4 influences the activation of the transcription factor NF-κB, which triggers the generation of acute inflammatory responses. V. cholerae CheY4 was cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY4 grown in space group C2 diffracted to 1.67 Å resolution, with unit-cell parameters a = 94.4, b = 31.9, c = 32.6 Å, ß = 96.5°, whereas crystals grown in space group P3(2)21 diffracted to 1.9 Å resolution, with unit-cell parameters a = b = 56.104, c = 72.283 Å, γ = 120°.

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar 'switch complex' in Vibrio cholerae.

Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1-CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ;switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 A3 Da(-1) (47% solvent content) assuming the presence of one molecule per asymmetric unit.

Crystallization and preliminary X-ray analysis of Psu, an inhibitor of the bacterial transcription terminator Rho.

Psu, a coat protein from bacteriophage P4, inhibits Rho-dependent transcription termination both in vivo and in vitro. The Psu protein is alpha-helical in nature and appeared to be a dimer in solution. It interacts with Rho and affects the ATP binding and RNA-dependent ATPase activity of Rho, which in turn reduces the rate of RNA release from the elongation complex. Crystals of Psu were grown in space group I422 in the presence of PEG, with unit-cell parameters a = b = 148.76, c = 63.38 A and a calculated Matthews coefficient of 2.1 A(3) Da(-1) (41.5% solvent content), assuming the presence of two molecules in the asymmetric unit. A native data set was collected to 2.3 A resolution.

Synthesis and Biological Validation of a Harmine-Based, Central Nervous System (CNS)-Avoidant, Selective, Human ß-Cell Regenerative Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A (DYRK1A) Inhibitor.

Recently, our group identified that harmine is able to induce ß-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. Since, harmine suffers from a lack of selectivity, both against other kinases and CNS off-targets, we therefore sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity for off-targets while retaining human ß-cell proliferation activity. We carried out optimization of the 9-N-position of harmine to synthesize 29 harmine-based analogs. Several novel inhibitors showed excellent DYRK1A inhibition and human ß-cell proliferation capability. An optimized DYRK1A inhibitor, 2-2c, was identified as a novel, efficacious in vivo lead candidate. 2-2c also demonstrates improved selectivity for kinases and CNS off-targets, as well as in vivo efficacy for ß-cell proliferation and regeneration at lower doses than harmine. Collectively, these findings demonstrate that 2-2c is a much improved in vivo lead candidate as compared to harmine for the treatment of diabetes.

Inhibition and Crystal Structure of the Human DHTKD1-Thiamin Diphosphate Complex.

DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 A) and biochemical basis.

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu-->Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8x10(10) M(-1) which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 A) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.

Conformational barrier of CheY3 and inability of CheY4 to bind FliM control the flagellar motor action in Vibrio cholerae.

Vibrio cholerae contains multiple copies of chemotaxis response regulator (VcCheY1-VcCheY4) whose functions are elusive yet. Although previous studies suggested that only VcCheY3 directly switches the flagellar rotation, the involvement of VcCheY4 in chemotaxis could not be ruled out. None of these studies, however, focused on the structure, mechanism of activation or molecular basis of FliM binding of the VcCheYs. From the crystal structures of Ca(2+) and Mg(2+) bound VcCheY3 we proposed the presence of a conformational barrier composed of the hydrophobic packing of W61, M88 and V106 and a unique hydrogen bond between T90 and Q97 in VcCheY3. Lesser fluorescence quenching and higher Km value of VcCheY3, compared to its mutants VcCheY3-Q97A and VcCheY3-Q97A/E100A supported our proposition. Furthermore, aforesaid biochemical data, in conjunction with the structure of VcCheY3-Q97A, indicated that the coupling of T90 and Q97 restricts the movement of T90 toward the active site reducing the stabilization of the bound phosphate and effectively promoting autodephosphorylation of VcCheY3. The structure of BeF3(-) activated VcCheY3 insisted us to argue that elevated temperature and/or adequacy of phosphate pool might break the barrier of the free-state VcCheY3 and the conformational changes, required for FliM binding, occur upon phosphorylation. Structure of VcCheY4 has been solved in the free and sulfated states. VcCheY4(sulf), containing a bound sulfate at the active site, appears to be more compact and stable with a longer α4 helix, shorter ß4α4 loop and hydrogen bond between T82 and the sulfate compared to VcCheY4(free). While pull down assay of VcCheYs with VcFliMNM showed that only activated VcCheY3 can interact with VcFliMNM and VcCheY4 cannot, a knowledge based docking explained the molecular mechanism of the interactions between VcCheY3 and VcFliM and identified the limitations of VcCheY4 to interact with VcFliM even in its phosphorylated state.

The compact conformation of the Plasmodium knowlesi myosin tail interacting protein MTIP in complex with the C-terminal helix of myosin A.

The myosin motor of the malaria parasite's invasion machinery moves over actin fibers while it is making critical contacts with the myosin-tail interacting protein (MTIP). Previously, in a "compact" Plasmodium falciparum MTIP•MyoA complex, MTIP domains 2 (D2) and 3 (D3) make contacts with the MyoA helix, and the central helix is kinked, but in an "extended" Plasmodium knowlesi MTIP•MyoA complex only D3 interacts with the MyoA helix, and the central helix is fully extended. Here we report the crystal structure of the compact P. knowlesi MTIP•MyoA complex. It appears that, depending on the pH, P. knowlesi MTIP can adopt either the compact or the extended conformation to interact with MyoA. Only at pH values above ~7.0, can key hydrogen bonds can be formed by the imidazole group of MyoA His810 with an aspartate carboxylate from the hinge of MTIP and a lysine amino group of MyoA simultaneously.

The structure of the D3 domain of Plasmodium falciparum myosin tail interacting protein MTIP in complex with a nanobody.

Apicomplexan parasites enter host cells by many sophisticated steps including use of an ATP-powered invasion machinery. The machinery consists of multiple proteins, including a special myosin (MyoA) which moves along an actin fiber and which is connected to the myosin tail interaction protein (MTIP). Here we report a crystal structure of the major MyoA-binding domain (D3) of Plasmodium falciparum MTIP in complex with an anti-MTIP nanobody. In this complex, the MyoA-binding groove in MTIP-D3 is considerably less accessible than when occupied by the MyoA helix, due to a shift of two helices. The nanobody binds to an area slightly overlapping with the MyoA binding groove, covering a hydrophobic region next to the groove entrance. This provides a new avenue for arriving at compounds interfering with the invasion machinery since small molecules binding simultaneously to the nanobody binding site and the adjacent MyoA binding groove would prevent MyoA binding by MTIP.

Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors.

For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed "mini-proteins," containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECI(L)-WCI(S), ETI(L)-WCI(S), and STI(L)-WCI(S), where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECI(L)-WCI(S) and STI(L)-WCI(S) behave like good inhibitors, ETI(L)-WCI(S) behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETI(L)-WCI(S) shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETI(L)-WCI(S) leading to a loss of canonical conformation, explaining its substrate-like behavior. Incorporation of this barrier back in ETI(L)-WCI(S) through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop-scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.