AI-Based Protein Interaction Screening and Identification (AISID).

In this study, we presented an AISID method extending AlphaFold-Multimer's success in structure prediction towards identifying specific protein interactions with an optimized AISIDscore. The method was tested to identify the binding proteins in 18 human TNFSF (Tumor Necrosis Factor superfamily) members for each of 27 human TNFRSF (TNF receptor superfamily) members. For each TNFRSF member, we ranked the AISIDscore among the 18 TNFSF members. The correct pairing resulted in the highest AISIDscore for 13 out of 24 TNFRSF members which have known interactions with TNFSF members. Out of the 33 correct pairing between TNFSF and TNFRSF members, 28 pairs could be found in the top five (including 25 pairs in the top three) seats in the AISIDscore ranking. Surprisingly, the specific interactions between TNFSF10 (TNF-related apoptosis-inducing ligand, TRAIL) and its decoy receptors DcR1 and DcR2 gave the highest AISIDscore in the list, while the structures of DcR1 and DcR2 are unknown. The data strongly suggests that AlphaFold-Multimer might be a useful computational screening tool to find novel specific protein bindings. This AISID method may have broad applications in protein biochemistry, extending the application of AlphaFold far beyond structure predictions.

Crystal Structure of Major Envelope Protein VP24 from White Spot Syndrome Virus.

White spot syndrome virus (WSSV) is one of the major and most serious pathogen in the shrimp industry. As one of the most abundant envelope protein, VP24 acts as a core protein interacting with other structure proteins and plays an important role in virus assembly and infection. Here, we have presented the crystal structure of VP24 from WSSV. In the structure, VP24 consists of a nine-stranded ß-barrel fold with mostly antiparallel ß-strands, and the loops extending out the ß-barrel at both N-terminus and C-terminus, which is distinct to those of the other two major envelope proteins VP28 and VP26. Structural comparison of VP24 with VP26 and VP28 reveals opposite electrostatic surface potential properties of them. These structural differences could provide insight into their differential functional mechanisms and roles for virus assembly and infection. Moreover, the structure reveals a trimeric assembly, suggesting a likely natural conformation of VP24 in viral envelope. Therefore, in addition to confirming the evolutionary relationship among the three abundant envelope proteins of WSSV, our structural studies also facilitate a better understanding of the molecular mechanism underlying special roles of VP24 in WSSV assembly and infection.

Overexpression of Arabidopsis NIMIN1 results in salicylate intolerance.

The transcriptional regulator NPR1 mediates salicylic acid (SA)-induced plant immunity. NPR1 is also required for tolerance to high concentrations of SA. NPR1-interacting protein, NIMIN1, represses immune response by interacting with and negating NPR1. We tested the salicylic acid tolerance of transgenic plants overexpressing NIMIN1 and found that these plants displayed SA intolerance, similar to the npr1 mutant, due to sequestration of NPR1 by NIMIN1. Plants overexpressing mutated NIMIN1 that cannot interact with NPR1 showed no SA tolerance defect. Gene expression analysis showed that NPR1 is required for SA-stress induced as well as pathogen-induced NIMIN1 expression. These results indicate that over-accumulation of a negative regulator renders plants hypersensitive to SA by limiting NPR1 function. Furthermore, NPR1 activates negative regulators such as NIMIN1 for feedback inhibition of SA signaling to maintain immune homeostasis.

Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Structure of the Archaeoglobus fulgidus orphan ORF AF1382 determined by sulfur SAD from a moderately diffracting crystal.

The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using a moderately diffracting crystal and 1.9 Å wavelength synchrotron X-rays. AF1382 was selected as a structural genomics target by the Southeast Collaboratory for Structural Genomics (SECSG) since sequence analyses showed that it did not belong to the Pfam-A database and thus could represent a novel fold. The structure was determined by exploiting longer wavelength X-rays and data redundancy to increase the anomalous signal in the data. AF1382 is a 95-residue protein containing five S atoms associated with four methionine residues and a single cysteine residue that yields a calculated Bijvoet ratio (ΔF(anom)/F) of 1.39% for 1.9 Å wavelength X-rays. Coupled with an average Bijvoet redundancy of 25 (two 360° data sets), this produced an excellent electron-density map that allowed 69 of the 95 residues to be automatically fitted. The S-SAD model was then manually completed and refined (R = 23.2%, R(free) = 26.8%) to 2.3 Å resolution (PDB entry 3o3k). High-resolution data were subsequently collected from a better diffracting crystal using 0.97 Å wavelength synchrotron X-rays and the S-SAD model was refined (R = 17.9%, R(free) = 21.4%) to 1.85 Å resolution (PDB entry 3ov8). AF1382 has a winged-helix-turn-helix structure common to many DNA-binding proteins and most closely resembles the N-terminal domain (residues 1-82) of the Rio2 kinase from A. fulgidus, which has been shown to bind DNA, and a number of MarR-family transcriptional regulators, suggesting a similar DNA-binding function for AF1382. The analysis also points out the advantage gained from carrying out data reduction and structure determination on-site while the crystal is still available for further data collection.

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family.

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B18-85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B18-85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg44 and Tyr51 as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.

The crystal structure analysis of group B Streptococcus sortase C1: a model for the "lid" movement upon substrate binding.

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible "lid" anchored in the active site. However, the mechanistic details of the "lid" displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its "lid" mutant and proposed a role of the "lid" as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.

Membrane binding mechanism of yeast mitochondrial peripheral membrane protein TIM44.

The protein translocations across mitochondrial membranes are carried out by specialized complexes, the Translocase of Outer Membrane (TOM) and Translocase of Inner Membrane (TIM). TIM23 translocon is responsible for translocating the mitochondrial matrix proteins across the mitochondrial inner membrane. Tim44 is an essential, peripheral membrane protein in TIM23 complex. Tim44 is tightly associated with the inner mitochondrial membrane on the matrix side. The Tim44 C-Terminal Domain (CTD) functions as an Inner Mitochondrial Membrane (IMM) anchor that recruits the Presequence protein Associated Motor (PAM) to the TIM23 channel. Using X-ray crystallographic and biochemical data, we show that the N-terminal helices A1 and A2 of Tim44 - CTD are crucial for its membrane tethering function. Based on our data, we propose a model showing how the N-terminal A1 and A2 amphipathic helices can either expose their hydrophobic face during membrane binding or conceal it in the soluble form. Therefore, the A1 and A2 helices of Tim44 may function as a membrane sensor.

Preliminary X-ray crystallographic studies of yeast Get3.

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus. The post-translational insertion of TA proteins into the ER membrane requires the cooperation of the Golgi ER-trafficking (GET) complex, which contains Get1, Get2 and Get3. Get3 is a cytosolic ATPase which can recognize and bind the TMD of the TA proteins. Get1 and Get2 are ER transmembrane proteins which can recruit and form a complex with TA-bound Get3. The GET complex carries out an energy-dependent process that facilitates the insertion of the TA-protein TMD into the ER membrane. In order to investigate the mechanism by which the GET complex functions to promote protein insertion into the ER membrane, yeast Get3 has been crystallized. The crystals diffracted to 2.7 A resolution using a synchrotron X-ray source. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 220.26, b = 112.95, c = 48.27 A. There is one Get3 dimer in the asymmetric unit, which corresponds to a solvent content of approximately 65%.

Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase.

Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between beta3-2 and beta3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.

The crystal structure of the putative peptide-binding fragment from the human Hsp40 protein Hdj1.

BACKGROUND: The mechanism by which Hsp40 and other molecular chaperones recognize and interact with non-native polypeptides is a fundamental question. How Hsp40 co-operates with Hsp70 to facilitate protein folding is not well understood. To investigate the mechanisms, we determined the crystal structure of the putative peptide-binding fragment of Hdj1, a human member of the type II Hsp40 family. RESULTS: The 2.7A structure reveals that Hdj1 forms a homodimer in the crystal by a crystallographic two-fold axis. The Hdj1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. When compared with another Hsp40 Sis1 structure, the domain I of Hdj1 is rotated by 7.1 degree from the main body of the molecule, which makes the cleft between the two Hdj1 monomers smaller that that of Sis1. CONCLUSION: This structural observation indicates that the domain I of Hsp40 may possess significant flexibility. This flexibility may be important for Hsp40 to regulate the size of the cleft. We propose an "anchoring and docking" model for Hsp40 to utilize the flexibility of domain I to interact with non-native polypeptides and transfer them to Hsp70.

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica.

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.

Crystal structure of yeast mitochondrial peripheral membrane protein Tim44p C-terminal domain.

The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of TIM23 translocon. Tim44p can tightly associate with the inner mitochondrial membrane. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, we have determined the crystal structure of the yeast Tim44p C-terminal domain to 3.2A resolution using the MAD method. The Tim44p C-terminal domain forms a monomer in the crystal structure and contains six alpha-helices and four antiparallel beta-strands. A large hydrophobic pocket was identified on the Tim44p structure surface. The N-terminal helix A1 is positively charged and the helix A1 protrudes out from the Tim44p main body.

The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40.

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms beta-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other.

Monitoring the anomalous scattering signal and noise levels in X-ray diffraction of crystals.

A statistical index R(as) is proposed in order to monitor the overall signal-to-noise ratio in an anomalous scattering data set. In this approach, symmetry-equivalent reflections are merged and grouped into centric and non-centric subsets. Reflections in the centric subset, which in theory should be equal, are used to estimate the noise level in the data. This approach differs from that used by most data-processing programs, in which the centric reflections are merged and averaged. By preserving the differences in centric reflections during data processing, an internal measure of the noise level can be estimated and used to analyze the quality of the anomalous signal in the data. An index R(as) is defined as the ratio of the average Bijvoet difference of merged acentric reflections to merged centric reflections. Test results on a variety of data show that R(as) has good correlation with the capability to determine the anomalous scattering substructure from the data. R(as) can also be useful in monitoring the quality of the data in terms of the data-collection strategy, instrument settings and data-processing software used. R(as) analysis has been implemented in the program 3DSCALE as part of a data-processing program suite that is under development in our laboratory.