PPI-HotspotDB: Database of Protein-Protein Interaction Hot Spots.

Single-point mutations of certain residues (so-called hot spots) impair/disrupt protein-protein interactions (PPIs), leading to pathogenesis and drug resistance. Conventionally, a PPI-hot spot is identified when its replacement decreased the binding free energy significantly, generally by ≥2 kcal/mol. The relatively few mutations with such a significant binding free energy drop limited the number of distinct PPI-hot spots. By defining PPI-hot spots based on mutations that have been manually curated in UniProtKB to significantly impair/disrupt PPIs in addition to binding free energy changes, we have greatly expanded the number of distinct PPI-hot spots by an order of magnitude. These experimentally determined PPI-hot spots along with available structures have been collected in a database called PPI-HotspotDB. We have applied the PPI-HotspotDB to create a nonredundant benchmark, PPI-Hotspot+PDBBM, for assessing methods to predict PPI-hot spots using the free structure as input. PPI-HotspotDB will benefit the design of mutagenesis experiments and development of PPI-hot spot prediction methods. The database and benchmark are freely available at https://ppihotspot.limlab.dnsalias.org.

Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

Soluble klotho (sKlotho), the shed ectodomain of α-klotho, protects the heart by down-regulating transient receptor potential canonical isoform 6 (TRPC6)-mediated calcium signaling. Binding to α2-3-sialyllactose moiety of gangliosides in lipid rafts and inhibition of raft-dependent signaling underlies the mechanism. A recent 3-Å X-ray structure of sKlotho in complex with fibroblast growth factor receptor (FGFR) and fibroblast growth factor 23 (FGF23) indicates that its ß6α6 loop might block access to the proposed binding site for α2-3-sialyllactose. It was concluded that sKlotho only functions in complex with FGFR and FGF23 and that sKlotho's pleiotropic effects all depend on FGF23. Here, we report that sKlotho can inhibit TRPC6 channels expressed in cells lacking endogenous FGFRs. Structural modeling and molecular docking show that a repositioned ß6α6 loop allows sKlotho to bind α2-3-sialyllactose. Molecular dynamic simulations further show the α2-3-sialyllactose-bound sKlotho complex to be stable. Domains mimicking sKlotho's sialic acid-recognizing activity inhibit TRPC6. The results strongly support the hypothesis that sKlotho can exert effects independent of FGF23 and FGFR.-Wright, J. D., An, S.-W., Xie, J., Lim, C., Huang, C.-L. Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn2+ Ligands and Roles in Damage Recognition and Repair.

The DNA base excision repair (BER) glycosylase MUTYH prevents DNA mutations by catalyzing adenine (A) excision from inappropriately formed 8-oxoguanine (8-oxoG):A mismatches. The importance of this mutation suppression activity in tumor suppressor genes is underscored by the association of inherited variants of MUTYH with colorectal polyposis in a hereditary colorectal cancer syndrome known as MUTYH-associated polyposis, or MAP. Many of the MAP variants encompass amino acid changes that occur at positions surrounding the two-metal cofactor-binding sites of MUTYH. One of these cofactors, found in nearly all MUTYH orthologs, is a [4Fe-4S]2+ cluster coordinated by four Cys residues located in the N-terminal catalytic domain. We recently uncovered a second functionally relevant metal cofactor site present only in higher eukaryotic MUTYH orthologs: a Zn2+ ion coordinated by three Cys residues located within the extended interdomain connector (IDC) region of MUTYH that connects the N-terminal adenine excision and C-terminal 8-oxoG recognition domains. In this work, we identified a candidate for the fourth Zn2+ coordinating ligand using a combination of bioinformatics and computational modeling. In addition, using in vitro enzyme activity assays, fluorescence polarization DNA binding assays, circular dichroism spectroscopy, and cell-based rifampicin resistance assays, the functional impact of reduced Zn2+ chelation was evaluated. Taken together, these results illustrate the critical role that the "Zn2+ linchpin motif" plays in MUTYH repair activity by providing for proper engagement of the functional domains on the 8-oxoG:A mismatch required for base excision catalysis. The functional importance of the Zn2+ linchpin also suggests that adjacent MAP variants or exposure to environmental chemicals may compromise Zn2+ coordination, and ability of MUTYH to prevent disease.

Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Structural and Physical Basis for Anti-IgE Therapy.

Omalizumab, an anti-IgE antibody, used to treat severe allergic asthma and chronic idiopathic urticaria, binds to IgE in blood or membrane-bound on B lymphocytes but not to IgE bound to its high (FcεRI) or low (CD23) affinity receptor. Mutagenesis studies indicate overlapping FcεRI and omalizumab-binding sites in the Cε3 domain, but crystallographic studies show FcεRI and CD23-binding sites that are far apart, so how can omalizumab block IgE from binding both receptors? We report a 2.42-Šomalizumab-Fab structure, a docked IgE-Fc/omalizumab-Fab structure consistent with available experimental data, and the free energy contributions of IgE residues to binding omalizumab, CD23, and FcεRI. These results provide a structural and physical basis as to why omalizumab cannot bind receptor-bound IgE and why omalizumab-bound IgE cannot bind to CD23/FcεRI. They reveal the key IgE residues and their roles in binding omalizumab, CD23, and FcεRI.

Identifying RNA-binding residues based on evolutionary conserved structural and energetic features.

Increasing numbers of protein structures are solved each year, but many of these structures belong to proteins whose sequences are homologous to sequences in the Protein Data Bank. Nevertheless, the structures of homologous proteins belonging to the same family contain useful information because functionally important residues are expected to preserve physico-chemical, structural and energetic features. This information forms the basis of our method, which detects RNA-binding residues of a given RNA-binding protein as those residues that preserve physico-chemical, structural and energetic features in its homologs. Tests on 81 RNA-bound and 35 RNA-free protein structures showed that our method yields a higher fraction of true RNA-binding residues (higher precision) than two structure-based and two sequence-based machine-learning methods. Because the method requires no training data set and has no parameters, its precision does not degrade when applied to 'novel' protein sequences unlike methods that are parameterized for a given training data set. It was used to predict the 'unknown' RNA-binding residues in the C-terminal RNA-binding domain of human CPEB3. The two predicted residues, F430 and F474, were experimentally verified to bind RNA, in particular F430, whose mutation to alanine or asparagine nearly abolished RNA binding. The method has been implemented in a webserver called DR_bind1, which is freely available with no login requirement at http://drbind.limlab.ibms.sinica.edu.tw.

Protein-Protein Docking Using EMAP in CHARMM and Support Vector Machine: Application to Ab/Ag Complexes.

In this work, we have (i) evaluated the ability of the EMAP method implemented in the CHARMM program to generate the correct conformation of Ab/Ag complex structures and (ii) developed a support vector machine (SVM) classifier to detect native conformations among the thousands of refined Ab/Ag configurations using the individual components of the binding free energy based on a thermodynamic cycle as input features in training the SVM. Tests on 24 Ab/Ag complexes from the protein-protein docking benchmark version 3.0 showed that based on CAPRI evaluation criteria, EMAP could generate medium-quality native conformations in each case. Furthermore, the SVM classifier could rank medium/high-quality native conformations mostly in the top six among the thousands of refined Ab/Ag configurations. Thus, Ab-Ag docking can be performed using different levels of protein representations, from grid-based (EMAP) to polar hydrogen (united-atom) to all-atom representation within the same program. The scripts used and the trained SVM are available at the www.charmm.org forum script repository.

DR_bind: a web server for predicting DNA-binding residues from the protein structure based on electrostatics, evolution and geometry.

DR_bind is a web server that automatically predicts DNA-binding residues, given the respective protein structure based on (i) electrostatics, (ii) evolution and (iii) geometry. In contrast to machine-learning methods, DR_bind does not require a training data set or any parameters. It predicts DNA-binding residues by detecting a cluster of conserved, solvent-accessible residues that are electrostatically stabilized upon mutation to Asp(-)/Glu(-). The server requires as input the DNA-binding protein structure in PDB format and outputs a downloadable text file of the predicted DNA-binding residues, a 3D visualization of the predicted residues highlighted in the given protein structure, and a downloadable PyMol script for visualization of the results. Calibration on 83 and 55 non-redundant DNA-bound and DNA-free protein structures yielded a DNA-binding residue prediction accuracy/precision of 90/47% and 88/42%, respectively. Since DR_bind does not require any training using protein-DNA complex structures, it may predict DNA-binding residues in novel structures of DNA-binding proteins resulting from structural genomics projects with no conservation data. The DR_bind server is freely available with no login requirement at http://dnasite.limlab.ibms.sinica.edu.tw.

Redesign of high-affinity nonspecific nucleases with altered sequence preference.

It is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3'O-side after thymine and adenine; i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage.

Combined experimental and theoretical study of long-range interactions modulating dimerization and activity of yeast geranylgeranyl diphosphate synthase.

We present here how two amino acid residues in the first helix distal from the main dimer interface modulate the dimerization and activity of a geranylgeranyl diphosphate synthase (GGPPs). The enzyme catalyzes condensation of farnesyl diphosphate and isopentenyl diphosphate to generate a C(20) product as a precursor for chlorophylls, carotenoids, and geranylgeranylated proteins. The 3D structure of GGPPs from Saccharomyces cerevisiae reveals an unique positioning of the N-terminal helix A, which protrudes into the other subunit and stabilizes dimerization, although it is far from the main dimer interface. Through a series of mutants that were characterized by analytic ultracentrifugation (AUC), the replacement of L8 and I9 at this helix with Gly was found sufficient to disrupt the dimer into a monomer, leading to at least 10(3)-fold reduction in activity. Molecular dynamics simulations and free energy decomposition analyses revealed the possible effects of the mutations on the protein structures and several critical interactions for maintaining dimerization. Further site-directed mutagenesis and AUC studies elucidated the molecular mechanism for modulating dimerization and activity by long-range interactions.

Mechanism of DNA-binding loss upon single-point mutation in p53.

Over 50% of all human cancers involve p53 mutations,which occur mostly in the sequence-specific DNA-binding central domain (p53c), yielding little/non-detectable af?nity to the DNA consensus site. Despite our current understanding of protein-DNA recognition,the mechanism(s) underlying the loss in protein-DNA binding afnity/ specificity upon single-point mutation are not well understood. Our goal is to identify the common factors governing the DNA-binding loss of p53c upon substitution of Arg 273 to His or Cys,which are abundant in human tumours. By computing the free energies of wild-type and mutant p53c binding to DNA and decomposing them into contributions from individual residues, the DNA-binding loss upon charge/noncharge -conserving mutation of Arg 273 was attributed not only to the loss of DNA phosphate contacts, but also to longer-range structural changes caused by the loss of the Asp 281 salt-bridge. The results herein and in previous works suggest that Asp 281 plays a critical role in the sequence-specific DNA-binding function of p53c by (i)orienting Arg 273 and Arg 280 in an optimal position to interact with the phosphate and base groups of the consensus DNA, respectively, and (ii) helping to maintain the proper DNA-binding protein conformation.

Factors governing loss and rescue of DNA binding upon single and double mutations in the p53 core domain.

The mutation of R273-->H in the p53 core domain (p53-CD) is one of the most common mutations found in human cancers. Although the 273H p53-CD retains the wild-type conformation and stability, it lacks sequence-specific DNA binding, a transactivation function and growth suppression. However, mutating T284-->R in the 273H p53-CD restores the DNA binding affinity, and transactivation and tumour suppressor functions. Since X-ray/NMR structures of DNA-free or DNA-bound mutant p53-CD molecules are unavailable, the factors governing the loss and rescue of sequence-specific DNA binding in the 273H and 273H+284R p53-CD, respectively, are unclear. Hence, we have carried out molecular dynamics (MD) simulations of the wild-type, single mutant and double mutant p53-CD, free and DNA bound, in the presence of explicit water molecules. Based on the MD structures, the DNA-binding free energy of each p53 molecule has been computed and decomposed into component energies and contributions from the interface residues. The wild-type and mutant p53-CD MD structures were found to be consistent with the antibody-binding, X-ray and NMR data. The predicted DNA binding affinity and specificity of both mutant p53-CDs were also in accord with experimental data. The non-detectable DNA binding of the 273H p53-CD is due mainly to the disruption of a hydrogen-bonding network involving R273, D281 and R280, leading to a loss of major groove binding by R280 and K120. The restoration of DNA binding affinity and specificity of the 273H+284R p53-CD is due mainly to the introduction of another DNA-binding site at position 284, leading to a recovery of major groove binding by R280 and K120. The important role of water molecules and the DNA major groove conformation as well as implications for structure-based linker rescue of the 273H p53-CD DNA-binding affinity are discussed.