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At least 433 records · Page 24Linked to original sources
PubMed2005

Structure of the apoptotic protease-activating factor 1 bound to ADP.

Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.

Adenosine Diphosphate↗
PubMed2006

Structural insights into the Notch-modifying glycosyltransferase Fringe.

Fringe proteins are beta1,3-N-acetylglucosaminyltransferases that modify Notch receptors, altering their ligand-binding specificity to regulate Notch signaling in development. We present the crystal structure of mouse Manic Fringe bound to UDP and manganese. The structure reveals amino acid residues involved in recognition of donor substrates and catalysis, and a putative binding pocket for acceptor substrates. Mutations of several invariant residues in this pocket impair Fringe activity in vivo.

Animals↗
PubMed2004

Structural and functional studies of mutations affecting the UBA domain of SQSTM1 (p62) which cause Paget's disease of bone.

Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Paget's disease of bone. The missense mutations resolve into those which retain [P392L (Pro(392)-->Leu), G411S] or abolish (M404V, G425R) the ability of the isolated UBA domain to bind Lys-48-linked polyubiquitin. These effects can be rationalized with reference to the solution structure of the UBA domain, which we have determined by NMR spectroscopy. The UBA domain forms a characteristic compact three-helix bundle, with a hydrophobic patch equivalent to that previously implicated in ubiquitin binding by other UBA domains. None of the mutations affect overall folding of the UBA domain, but both M404V and G425R involve residues in the hydrophobic patch, whereas Pro-392 and Gly-411 are more remote. A simple model assuming the isolated UBA domain is functioning as a compact monomer can explain the effects of the mutations on polyubiquitin binding. The P392L and G411S mutations do however have subtle local effects on secondary structure, which may become more relevant in full-length SQSTM1. Identification of the in vivo ubiquitylated substrates of SQSTM1 will be most informative in determining the functional significance of the SQSTM1-ubiquitin interaction, and consequences of the disease-associated mutations.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Inference of functional regions in proteins by quantification of evolutionary constraints.

Likelihood estimates of local rates of evolution within proteins reveal that selective constraints on structure and function are quantitatively stable over billions of years of divergence. The stability of constraints produces an intramolecular clock that gives each protein a characteristic pattern of evolutionary rates along its sequence. This pattern allows the identification of constrained regions and, because the rate of evolution is a quantitative measure of the strength of the constraint, of their functional importance. We show that results from such analyses, which require only sequence alignments, are consistent with experimental and mutational data. The methodology has significant predictive power and may be used to guide structure--function studies for any protein represented by a modest number of homologs in sequence databases.

Animals↗
PubMed2002

Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling.

Interleukin (IL)-1 plays an important role in inflammation and regulation of immune responses. The activated IL-1 receptor complex, which consists of the IL-1 receptor type I and the IL-1 receptor accessory protein (IL-1RAcP), generates multiple cellular responses including NF-kappaB activation, IL-2 secretion, and IL-2 promoter activation. Reconstitution experiments in EL4D6/76 cells lacking IL-1RAcP expression and IL-1 responsiveness were used to analyze structure-function relationships of the IL-1RAcP cytoplasmic tail. Mutating a potential tyrosine kinase phosphorylation motif and various conserved amino acid (aa) residues had no effect on IL-1 responsiveness. Truncation analyses revealed that box 3 of the TIR domain was required for NF-kappaB activation, IL-2 production, and c-Jun N-terminal kinase (JNK) activation, whereas IL-2 promoter activation was only partially inhibited. Surprisingly, deletion of aa 527-534 resulted in almost complete loss of all IL-1 responsiveness. Replacement of these aa with alanyl residues did not reconstitute NF-kappaB activation, IL-2 production, or JNK activation but partly restored IL-2 promoter activation. Immunoprecipitation data revealed a strong correlation between MyD88 binding with NF-kappaB activation and IL-2 production but not with IL-2 promoter activation. Taken together, our data indicate that box 3 of IL-1RAcP is critical for IL-1-dependent NF-kappaB activation and stabilization of IL-2 mRNA via JNK, whereas aa 527-534 largely contribute to IL-2 promoter activation.

Amino Acid Sequence↗
PubMed2004

The C-terminal residues in the alpha-interacting domain (AID) helix anchor CaV beta subunit interaction and modulation of CaV2.3 channels.

The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca(2+) channel alpha1 subunits binds with high affinity to Ca(V)beta auxiliary subunits. The recently solved crystal structures of the AID-Ca(V)beta complex in Ca(V)1.1/1.2 have revealed that this interaction occurs through a set of six mostly invariant residues Glu/Asp(6), Leu(7), Gly(9), Tyr(10), Trp(13), and Ile(14) (where the superscript refers to the position of the residue starting with the QQ signature doublet) distributed among three alpha-helical turns in the proximal section of the I-II linker. We show herein that alanine mutations of N-terminal AID residues Gln(1), Gln(2), Ile(3), Glu(4), Glu(6), Leu(7), and Gly(9) in Ca(V)2.3 did not abolish [(35)S]Ca(V)beta 1b or [(35)S]Ca(V)beta 3 subunit overlay binding to fusion proteins nor did they prevent the typical modulation of whole cell currents by Ca(V)beta 3. Mutations of the invariant Tyr(10) with either hydrophobic (Ala), aromatic (Phe), or positively charged (Arg, Lys) residues yielded Ca(V)beta 3-responsive whole cell currents, whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca(V)beta 3 binding and modulation. In contrast, modulation and binding by Ca(V)beta 3 was significantly weakened in I14A (neutral and hydrophobic) and I14S (neutral and polar) mutants and eradicated in negatively charged I14D and I14E or positively charged I14R and I14K mutants. Ca(V)beta 3-induced modulation was only preserved with the conserved I14L mutation. Molecular replacement analyses carried out using a three-dimensional homology model of the AID helix from Ca(V)2.3 suggests that a high degree of hydrophobicity and a restrained binding pocket could account for the strict structural specificity of the interaction site found at position Ile(14). Altogether these results indicate that the C-terminal residues Trp(13) (1) and Ile(14) anchor Ca(V)beta subunit functional modulation of HVA Ca(2+) channels.

Alanine↗
PubMed2006

Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition.

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-A resolution, respectively. The enzyme is composed of four domains: an N-terminal beta-domain (Met(1)-Asp(193)), a catalytic domain (Phe(194)-Gly(444)), a middle beta-domain (Thr(445)-Trp(546)), and a C-terminal alpha-domain (Ser(547)-Ala(870)). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX(18)E) is found in the domain. The zinc ion is coordinated by His(297), His(301), Glu(320), and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal alpha-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal alpha-domain. The N terminus of bestatin is recognized by Glu(121) and Glu(264), which are located in the N-terminal and catalytic domains, respectively. Glu(298) and Tyr(381), located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met(260) functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.

Amino Acid Sequence↗
PubMed2000

The interaction of drugs with DNA gyrase: a model for the molecular basis of quinolone action.

DNA gyrase supercoils DNA in bacteria. The fact that it is essential in all bacteria and absent from eukaryotes makes it an ideal drug target. We discuss the action of coumarin and quinolone drugs on gyrase. In the case of coumarins, the drugs are known to be competitive inhibitors of the gyrase ATPase reaction. From a combination of structural and biochemical studies, the molecular details of the gyrase-coumarin complex are well established. In the case of quinolones, the drugs are thought to act by stabilising a cleavage complex between gyrase and DNA that arrests polymerases in vivo. The exact nature of the gyrase-quinolone-DNA complex is not known; we propose a model for this complex based on structural and biochemical data.

Anti-Infective Agents↗
PubMed2006

TIP-1 has PDZ scaffold antagonist activity.

PDZ proteins usually contain multiple protein-protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand.

Animals↗
PubMed2002

ASPD (Artificially Selected Proteins/Peptides Database): a database of proteins and peptides evolved in vitro.

ASPD is a new curated database that incorporates data on full-length proteins, protein domains and peptides that were obtained through in vitro directed evolution processes (mainly by means of phage display). At present, the ASPD database contains data on 195 selection experiments, which were described in 112 original papers. For each experiment, the following information is given: (i) description of the target for binding, (ii) description of the protein or peptide which serves as the template for library construction and description of the native protein which binds the target, (iii) links to the major proteomic databases (SWISS-PROT, PDB, PROSITE and ENZYME), (iv) keywords referring to the biological significance of the experiment, (v) aligned sequences of proteins or peptides retrieved through in vitro evolution and relevant native or constructed sequences, (vi) the number of rounds of selection/amplification and (vii) the number of occurrences of clones with each sequence. The literature data include a full reference, a link to the MEDLINE database and the name of the corresponding author with his email address. ASPD has a user-friendly interface which allows for simple queries using the names of proteins and ligands, as well as keywords describing the biological role of the interaction studied, and also for queries based on authors' names. It is also possible to access the database by means of the SRS system, allowing complex queries. There is a BLAST search tool against the ASPD for looking directly for homologous sequences. Research tools of the ASPD allow the analysis of pairwise correlations in the sequences of proteins and peptides selected against one target. The URL for the ASPD database is http://www.sgi.sscc.ru/mgs/gnw/aspd/.

Animals↗
PubMed2002

PASS2: a semi-automated database of protein alignments organised as structural superfamilies.

PASS2 is a nearly automated version of CAMPASS and contains sequence alignments of proteins grouped at the level of superfamilies. This database has been created to fall in correspondence with SCOP database (1.53 release) and currently consists of 110 multi-member superfamilies and 613 superfamilies corresponding to single members. In multi-member superfamilies, protein chains with no more than 25% sequence identity have been considered for the alignment and hence the database aims to address sequence alignments which represent 26 219 protein domains under the SCOP 1.53 release. Structure-based sequence alignments have been obtained by COMPARER and the initial equivalences are provided automatically from a MALIGN alignment and subsequently augmented using STAMP4.0. The final sequence alignments have been annotated for the structural features using JOY4.0. Several interesting links are provided to other related databases and genome sequence relatives. Availability of reliable sequence alignments of distantly related proteins, despite poor sequence identity and single-member superfamilies, permit better sampling of structures in libraries for fold recognition of new sequences and for the understanding of protein structure-function relationships of individual superfamilies. The database can be queried by keywords and also by sequence search, interfaced by PSI-BLAST methods. Structure-annotated sequence alignments and several structural accessory files can be retrieved for all the superfamilies including the user-input sequence. The database can be accessed from http://www.ncbs.res.in/%7Efaculty/mini/campass/pass.html.

Amino Acid Sequence↗
PubMed2006

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana.

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.

Algal Proteins↗
PubMed1991

Influences of structural factors of biological media on the acoustic nonlinearity parameter B/A.

The influence of structural factors of biological media on the acoustic nonlinearity parameter B/A have been studied at the tissue, cellular, and molecular levels, using the thermodynamic and finite amplitude methods. B/A was determined as the structural factors of the media were altered physically and biochemically, while chemical composition was maintained unchanged. Significant structural dependencies of B/A were observed at all three levels; 26% of the dry weight contribution to the total B/A (the B/A value with water contribution subtracted) is due to the cell-cell adhesive force in liver tissue, 20% is due to the hepatocyte cellular structure, and 15% is due to secondary and tertiary protein structure.

Acoustics↗
PubMed2000

Design of a novel membrane-destabilizing peptide selectively acting on acidic liposomes.

The design of amphipathic peptides resulted in a novel peptide with a selective ability to destabilize lipid bilayers of acidic liposomes. The newly synthesized peptide, termed mast 21, is a 21-residue long amino acid chain and can only act effectively on acidic liposomes lacking cholesterol. Moreover, mast 21 killed gram-positive and gram-negative bacteria, and it had no hemolytic activity. The antimicrobial and hemolytic activities paralleled the results of membrane destabilizing activity using liposomes. Circular dichroism and Trp-fluorescence emission spectra showed changes in the peptide conformation and circumstances around the peptide during interaction with liposomes. These changes were consistent with an increased alpha-helical content and a less polar environment for the tryptophan residue of the peptide. Mast 21 was observed under dark-field microscopy in real time attacking liposomes. Acidic liposomes were attacked, which resulted in peeling of the lipid bilayer with its subsequent destruction.

Amino Acid Sequence↗
PubMed2004

Involvement of a heptad repeat in the carboxyl terminus of the dihydropyridine receptor beta1a subunit in the mechanism of excitation-contraction coupling in skeletal muscle.

Chimeras consisting of the homologous skeletal dihydropyridine receptor (DHPR) beta1a subunit and the heterologous cardiac/brain beta2a subunit were used to determine which regions of beta1a were responsible for the skeletal-type excitation-contraction (EC) coupling phenotype. Chimeras were transiently transfected in beta1 knockout myotubes and then voltage-clamped with simultaneous measurement of confocal fluo-4 fluorescence. All chimeras expressed a similar density of DHPR charge movements, indicating that the membrane density of DHPR voltage sensors was not a confounding factor in these studies. The data indicates that a beta1a-specific domain present in the carboxyl terminus, namely the D5 region comprising the last 47 residues (beta1a 478-524), is essential for expression of skeletal-type EC coupling. Furthermore, the location of beta1aD5 immediately downstream from conserved domain D4 is also critical. In contrast, chimeras in which beta1aD5 was swapped by the D5 region of beta2a expressed Ca(2+) transients triggered by the Ca(2+) current, or none at all. A hydrophobic heptad repeat is present in domain D5 of beta1a (L478, V485, V492). To determine the role of this motif, residues in the heptad repeat were mutated to alanines. The triple mutant beta1a(L478A/V485A/V492A) recovered weak skeletal-type EC coupling (DeltaF/F(max) = 0.4 +/- 0.1 vs. 2.7 +/- 0.5 for wild-type beta1a). However, a triple mutant with alanine substitutions at positions out of phase with the heptad repeat, beta1a(S481A/L488A/S495A), was normal (DeltaF/F(max) = 2.1 +/- 0.4). In summary, the presence of the beta1a-specific D5 domain, in its correct position after conserved domain D4, is essential for skeletal-type EC coupling. Furthermore, a heptad repeat in beta1aD5 controls the EC coupling activity. The carboxyl terminal heptad repeat of beta1a might be involved in protein-protein interactions with ryanodine receptor type 1 required for DHPR to ryanodine receptor type 1 signal transmission.

Animals↗
PubMed2002

Electrostatics in protein binding and function.

Protein electrostatic properties stem from the proportion and distribution of polar and charged residues. Polar and charged residues regulate the electrostatic properties by forming short-range interactions, like salt-bridges and hydrogen-bonds, and by defining the over-all electrostatic environment in the protein. Electrostatics play a major role in defining the mechanisms of protein-protein complex formation, molecular recognitions, thermal stabilities, conformational adaptabilities and protein movements. For example:- Functional hinges, or flexible regions of the protein, lack short-range electrostatic interactions; Thermophilic proteins have higher electrostatic interactions than their mesophilic counter parts; Increase in binding specificity and affinity involve optimization of electrostatics; High affinity antibodies have higher, and stronger, electrostatic interactions with their antigens; Rigid parts of proteins have higher and stronger electrostatic interactions. In this review we address the significance of electrostatics in protein folding, binding and function. We discuss that the electrostatic properties are evolutionally selected by a protein to perform an specific function. We also provide bona fide examples to illustrate this. Additionally, using continuum electrostatic and molecular dynamics approaches we show that the "hot-spot" inter-molecular interactions in a very specific antibody-antigen binding are mainly established through charged residues. These "hot-spot" molecular interactions stay intact even during high temperature molecular dynamics simulations, while the other inter-molecular interactions, of lesser functional significance, disappear. This further corroborates the significance of charge-charge interactions in defining binding mechanisms. High affinity binding frequently involves "electrostatic steering". The forces emerge from over-all electrostatic complementarities and by the formation of charged and polar interactions. We demonstrate that although the high affinity binding of barnase-barstar and anti-hen egg white lysozyme (HEL) antibody-HEL complexes involve different molecular mechanisms, it is electrostatically regulated in both the cases. These observations, and several other studies, suggest that a fine tuning of local and global electrostatic properties are essential for protein binding and function.

Hydrogen Bonding↗
PubMed2005

Protein phosphorylation and signal transduction modulation: chemistry perspectives for small-molecule drug discovery.

Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.

Drug Design↗