Structure Of Core Domain Research Articles

The UBAP1 Subunit of ESCRT-I Interacts with Ubiquitin via a SOUBA Domain

SummaryThe endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.

Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis.

The spliceosome is a dynamic macromolecular machine that assembles on pre-mRNA substrates and catalyses the excision of non-coding intervening sequences (introns)1-3. Four of the five major components of the spliceosome, U1, U2, U4 and U5 snRNPs, contain seven Sm proteins (SmB/B’, SmD1, SmD2, SmD3, SmE, SmF and SmG) in common4,5. Following export of the U1, U2, U4 and U5 snRNAs to the cytoplasm6,7, the seven Sm proteins chaperoned by the survival of motor neurons (SMN) complex assemble around a single-stranded, U-rich sequence called the Sm site in each snRNA, to form the core domain of the respective snRNP particle8,9. Core domain formation is a prerequisite for re-import into the nucleus10, where these snRNPs mature via addition of their particle-specific proteins. Here we present a crystal structure of the U4 snRNP core domain at 3.6 Å resolution, detailing how the Sm site heptad (AUUUUUG) binds inside the central hole of the heptameric ring of Sm proteins, interacting one-to-one with SmE-SmG-SmD3-SmB-SmD1-SmD2-SmF. An irregular backbone conformation of the Sm site sequence combined with the asymmetric structure of the heteromeric protein ring allows each base to interact in a distinct manner with four key residues at equivalent positions in the L3 and L5 loops of the Sm fold. A comparison of this structure with the U1 snRNP at 5.5 Å resolution11,12 reveals snRNA-dependent structural changes outside the Sm fold, which may facilitate the binding of particle-specific proteins that is crucial to biogenesis of spliceosomal snRNPs.

Direct Activation of Human Phospholipase C by Its Well Known Inhibitor U73122

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Molecular characterization and structure analysis of RPL10/QM-like protein from the red drum Sciaenops ocellatus (Sciaenidae)

The QM-like gene encodes a ribosomal protein L10. Besides housekeeping roles in protein synthesis, QM-like proteins have multiple extraribosomal functions during cell growth, cell differentiation and apoptosis. We obtained the full-length cDNA of QM-like protein (designated as SoQM) from the salt water game fish Sciaenops ocellatus, using RACE-PCR. The sequence consists of 740 bp, encoding 215-amino acid residues with 24.60 kDa. The AA sequence of the SoQM protein contains a series of functional motifs that belong to the QM family signature, which is conserved among different species. The SoQM gene contains five introns and six exons. The expression pattern of SoQM as determined by RT-PCR indicated that SoQM mRNA was expressed in all tissues tested, including brain, gill, head-kidney, intestine, stomach, heart, spleen, blood, muscle, and gonads. The phylogenetic tree constructed with MEGA 4.0 showed that SoQM clusters together with that of other fish. It was found that the sequences of the SoQM gene are highly conserved, suggesting the fundamental and critical functions of SoQM in S. ocellatus. The three-dimensional structure of the SoQM protein core domain (4~169) was predicted by the Swiss-Model program. Compared with QM proteins in other species, the main structure of SoQM protein was conserved, while the C-terminal domain was different from other QM-like proteins. Prediction of the three-dimensional structure of SoQM would provide valuable insight into the molecular basis of protein function, allowing an effective design of experiments, such as site-directed mutagenesis, studies of disease-related mutations or structure-based design of specific inhibitors.

Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors. Structured summary MINT- 7713125: IN (uniprotkb: P04585) and IN (uniprotkb: P04585) bind (MI: 0407) by X-ray crystallography (MI: 0114)

Molecular cloning of the terminal complement components C6 and C8β of cartilaginous fish

The terminal complement components (TCCs) of mammals, C6, C7, C8α, C8β, and C9, are a group of serum proteins involved in the cytolytic killing of microbial pathogens. The mammalian TCCs share a unique core domain structure and were probably generated by the duplication of the ancestral TCC gene and subsequent addition and/or deletion of the N- and C-terminal domains. Proteins and genes for all the TCCs have been identified from bony fish. In contrast, no TCC gene has been identified from cyclostome lamprey using whole-genome shotgun-sequence analysis and liver EST analysis. To clarify the evolutionary origin of TCCs, we performed degenerate RT-PCR and RACE analyses of the cartilaginous fish liver and identified the C6 gene from a shark, Mustelus manazo, and the C8B gene from a chimaera, Chimaera phantasma. The presence of the C6 gene in shark suggests that one of the most crucial steps in the establishment of the cytolytic complement pathway, the addition of the FIM and CCP domains to the primitive TCC, occurred in a common ancestor of the jawed vertebrates. These results also indicate that the gene duplications among TCCs occurred at an early stage of the jawed vertebrate evolution.

Electrospray Ionization Mass Spectra of Dipeptide Derivatives

AbstractBased on the structure of the HIV integrase core domain, dipeptide derivatives, as a type of HIV integrase inhibitor, were synthesized, and their fragmentation pathways were investigated by electrospray ionization mass spectrometry (ESI‐MSn) in conjunction with tandem mass spectrometry (MS/MS). In order to better understand the fragmentation pathways, the MS2 and MS3 spectra of the title compound were obtained. The main fragmentation pathways occur by the cleavage of the C–CO bonds between N‐(benzothiazol‐2‐yl)aminocarbonyl and methylene, NH–CO bonds between the NH groups and carbonyl groups. Electrospray ionization was proven to be a good method for the structural characterization and identification of this kind of compound.

Interpreting a Low Resolution Map of Human U1 snRNP Using Anomalous Scatterers

SummaryWe recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 Å resolution electron density map. At 5–7 Å resolution, α helices and β sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 Å. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Crystal structure of the mouse p53 core domain in zinc‐free state

Crystal structure of the mouse p53 core domain in zinc‐free state

Study of the domain structure of a permalloy strip with uncharged domain walls

The decrease in the dimensions of the core of a magnetic head with increasing recording density leads to the necessity for the allowance of the changes in the micromagnetic structure of the magnetic core to be taken into account when calculating the writing field. A micromagnetic simulation of the process of changes in the magnetic structure under the action of an external magnetic field has been performed on the example of a rectangular magnetic core. The initial equilibrium periodic domain structure contains uncharged domain walls. When an external magnetic field acts along the longer side of the core, the structure changes so that the domain walls remain uncharged; i.e., no additional sources of stray field are generated, which simplifies the calculation of the domain structures of magnetic cores of a more complex shape. The thus-obtained parameters of the peripheral zone of the magnetic core make it possible to calculate the writing field and to choose optimum dimensions of the heads designed.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.

BackgroundTreatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs.MethodsPhylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR.ResultsThe calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810.ConclusionWe report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.

High-resolution structure of the p53 core domain: implications for binding small-molecule stabilizing compounds

The p53 transcriptional regulator is the most frequently mutated protein in human cancers and the majority of tumor-derived p53 mutations map to the central DNA-binding core domain, with a subset of these mutations resulting in reduced p53 stability. Here, the 1.55 A crystal structure of the mouse p53 core domain with a molecule of tris(hydroxymethyl)aminomethane (Tris) bound through multiple hydrogen bonds to a region of p53 shown to be important for repair of a subset of tumor-derived p53-stability mutations is reported. Consistent with the hypothesis that Tris binding stabilizes the p53 core domain, equilibrium denaturation experiments are presented that demonstrate that Tris binding increases the thermodynamic stability of the mouse p53 core domain by 3.1 kJ mol(-1) and molecular-dynamic simulations are presented revealing an overall reduction in root-mean-square deviations of the core domain of 0.7 A when Tris is bound. It is also shown that these crystals of the p53 core domain are suitable for the multiple-solvent crystal structure approach to identify other potential binding sites for possible core-domain stabilization compounds. Analysis of the residue-specific temperature factors of the high-resolution core-domain structure, coupled with a comparison with other core-domain structures, also reveals that the L1, H1-S5 and S7-S8 core-domain loops, also shown to mediate various p53 activities, harbor inherent flexibility, suggesting that these regions might be targets for other p53-stabilizing compounds. Together, these studies provide a molecular scaffold for the structure-based design of p53-stabilization compounds for development as possible therapeutic agents.

Novel epididymal protease inhibitors with Kazal or WAP family domain

The epididymal maturation of spermatozoa is regulated by changes in the luminal ion concentration and the processing of the sperm surface membrane by several glycosidases and proteases. In the present study, we identified five novel protease inhibitors that are highly expressed in the mouse epididymis. Four of the proteins were found to belong to the Kazal protease inhibitor family and were named SPINK8, SPINK10, SPINK11, and SPINK12, whereas one of the proteins, WFDC10, contained the WAP four-disulfide core domain structure. The novel genes showed very specific segmental expression patterns. The expression of all the five genes was regulated by testis-derived factors and decreased after gonadectomy. With the exception of Spink11, mRNA levels could be restored by testosterone replacement. We hypothesize that the protease inhibitors discovered represent a group of epididymal genes that contribute to the regulation of sperm maturation by regulating the proteolytic processing of the sperm membrane during epididymal transit.

ESCRT-I Core and ESCRT-II GLUE Domain Structures Reveal Role for GLUE in Linking to ESCRT-I and Membranes

ESCRT complexes form the main machinery driving protein sorting from endosomes to lysosomes. Currently, the picture regarding assembly of ESCRTs on endosomes is incomplete. The structure of the conserved heterotrimeric ESCRT-I core presented here shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/Tsg101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its "steadiness box" in the stability of ESCRT-I. We show that yeast ESCRT-I links directly to ESCRT-II, through a tight interaction of Vps28 (ESCRT-I) with the yeast-specific zinc-finger insertion within the GLUE domain of Vps36 (ESCRT-II). The crystal structure of the GLUE domain missing this insertion reveals it is a split PH domain, with a noncanonical lipid binding pocket that binds PtdIns3P. The simultaneous and reinforcing interactions of ESCRT-II GLUE domain with membranes, ESCRT-I, and ubiquitin are critical for ubiquitinated cargo progression from early to late endosomes.

Solution structure of p53 core domain: Structural basis for its instability

The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using state-of-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.

Structure and Function of the XpsE N-Terminal Domain, an Essential Component of the Xanthomonas campestris Type II Secretion System

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states. By mediating interaction between XpsE and the cytoplasmic membrane protein XpsL, XpsEN anchors XpsE to the membrane-associated secretion complex to allow the coupling between ATP utilization and exoprotein secretion. The structure of XpsEN observed in crystal form P4(3)2(1)2 is composed of a 90-residue alpha/beta sandwich core domain capped by a 62-residue N-terminal helical region. The core domain exhibits structural similarity with the NifU-like domain, suggesting that XpsE(N) may be involved in the regulation of XpsE ATPase activity. Surprisingly, although a similar core domain structure was observed in crystal form I4(1)22, the N-terminal 36 residues of the helical region undergo a large structural rearrangement. Deletion analysis indicates that these residues are required for exoprotein secretion by mediating the XpsE/XpsL interaction. Site-directed mutagenesis study further suggests the more compact conformation observed in the P4(3)2(1)2 crystal likely represents the XpsL binding-competent state. Based on these findings, we speculate that XpsE might function in T2SS by cycling between two conformational states. As a closely related protein to XpsE, secretion ATPase PilB may function similarly in the type IV pilus assembly.

Crystal Structures Representing the Michaelis Complex and the Thiouronium Reaction Intermediate of Pseudomonas aeruginosa Arginine Deiminase

L-arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys406, His278, Asp280, and Asp166. The function of Cys406 in nucleophilic catalysis has been demonstrated by transient kinetic studies. In this study, the structure of the C406A mutant in complex with L-arginine is reported to provide a snapshot of the enzyme.substrate complex. Through the comparison of the structures of apoenzyme and substrate-bound enzyme, a substrate-induced conformational transition, which might play an important role in activity regulation, was discovered. Furthermore, the position of the guanidinium group of the bound substrate relative to the side chains of His278, Asp280, and Asp166 indicated that these residues mediate multiple proton transfers. His278 and Asp280, which are positioned to activate the water nucleophile in the hydrolysis of the S-alkylthiouronium intermediate, were replaced with alanine to stabilize the intermediate for structure determination. The structures determined for the H278A and D280A mutants co-crystallized with L-arginine provide a snapshot of the S-alkylthiouronium adduct formed by attack of Cys406 on the guanidinium carbon of L-arginine followed by the elimination of ammonia. Asp280 and Asp166 engage in ionic interactions with the guanidinium group in the C406A ADI. L-arginine structure and might orient the reaction center and participate in proton transfer. Structure determination of D166A revealed the apoD166A ADI. The collection of structures is interpreted in the context of recent biochemical data to propose a model for ADI substrate recognition and catalysis.

Alanine Scanning Mutants of the HIV gp41 Loop

Based on mutagenesis and structural studies of human immunodeficiency virus (HIV) envelope proteins, the loop region of gp41 is thought to directly interact with gp120. The importance of the HIV gp41 loop region to envelope function has been systematically examined by alanine scanning of all gp41 loop residues and the subsequent characterization of the mutagenic effects on viral entry, envelope expression, envelope processing, and gp120 association with gp41. With respect to the wild-type gp41, mutational effects on viral entry fall into four classes as follows: 1) little or no effect (G594A, S599A, G600A, K601A, N611A, S615A, N616A, and L619A); 2) significantly reduced entry (I595A, L602A, I603A, V608A, and K617A); 3) abolished entry (L593A, W596A, G597A, T606A, W610A, W614A, S618A, and I622A); and 4) enhanced entry (T605A, P609A, S613A, E620A, and Q621A). The reduced functionality of many mutants was apparently due to either disruption of envelope processing (L593A and T606A), viral incorporation of the envelope (W610A, W614A, and I662A), or increased dissociation of gp120 (W596A, G597A, and S618A). The extreme sensitivity of the gp120-gp41 interaction to alanine substitutions (e.g. the G597A and S618A mutants are relatively conservative substitutions) suggests that this association is an attractive and novel target for future drug discovery efforts.

Crystal structure of a predicted phosphoribosyltransferase (TT1426) from Thermus thermophilus HB8 at 2.01 Å resolution

TT1426, from Thermus thermophilus HB8, is a conserved hypothetical protein with a predicted phosphoribosyltransferase (PRTase) domain, as revealed by a Pfam database search. The 2.01 A crystal structure of TT1426 has been determined by the multiwavelength anomalous dispersion (MAD) method. TT1426 comprises a core domain consisting of a central five-stranded beta sheet surrounded by four alpha-helices, and a subdomain in the C terminus. The core domain structure resembles those of the type I PRTase family proteins, although a significant structural difference exists in an inserted 43-residue region. The C-terminal subdomain corresponds to the "hood," which contains a substrate-binding site in the type I PRTases. The hood structure of TT1426 differs from those of the other type I PRTases, suggesting the possibility that TT1426 binds an unknown substrate. The structure-based sequence alignment provides clues about the amino acid residues involved in catalysis and substrate binding.

Domain structure in Joule-heated CoFeSiB glass-covered amorphous microwires probed by magnetoimpedance and ferromagnetic resonance

Impedance spectra (100 kHz⩽f⩽1.8 GHz) were measured at different magnetic fields, and magnetoimpedance and magnetization measurements were performed in as-produced and Joule-heated Co70.4Fe4.6Si10B15 glass-covered amorphous microwires. From the magnetization curves and ferromagnetic resonance features obtained from the impedance spectra, it was possible to propose a domain structure in the as-produced microwires, as well as to follow its evolution with the annealing current. The inner core and outer shell domain structure were observed. The outer shell domains evolve from a circumferential, in the as-cast sample, to a helical structure in the case of the annealed samples. On the other hand, the inner core evolves from a domain structure typical of a radial anisotropy to a longitudinal one. This domain structure evolution is explained in terms of the combined effects of the stress, promoted by the annealing temperature, and the magnetic field caused by the annealing current.