Biomacromolecular interactions, assemblies and machines: a structural view.
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Crystallography, X-Ray↗
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Results for “Protein Structure, Tertiary”
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Mutations in the Norrie disease gene.
We report our experience to date in mutation identification in the Norrie disease (ND) gene. We carried out mutational analysis in 26 kindreds in an attempt to identify regions presumed critical to protein function and potentially correlated with generation of the disease phenotype. All coding exons, as well as noncoding regions of exons 1 and 2, 636 nucleotides in the noncoding region of exon 3, and 197 nucleotides of 5' flanking sequence, were analyzed for single-strand conformation polymorphisms (SSCP) by polymerase chain reaction (PCR) amplification of genomic DNA. DNA fragments that showed altered SSCP band mobilities were sequenced to locate the specific mutations. In addition to three previously described submicroscopic deletions encompassing the entire ND gene, we have now identified 6 intragenic deletions, 8 missense (seven point mutations, one 9-bp deletion), 6 nonsense (three point mutations, three single bp deletions/frameshift) and one 10-bp insertion, creating an expanded repeat in the 5' noncoding region of exon 1. Thus, mutations have been identified in a total of 24 of 26 (92%) of the kindreds we have studied to date. With the exception of two different mutations, each found in two apparently unrelated kindreds, these mutations are unique and expand the genotype database. Localization of the majority of point mutations at or near cysteine residues, potentially critical in protein tertiary structure, supports a previous protein model for norrin as member of a cystine knot growth factor family (Meitinger et al., 1993). Genotype-phenotype correlations were not evident with the limited clinical data available, except in the cases of larger submicroscopic deletions associated with a more severe neurologic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four Vlambda6 proteins.
Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (VL) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the VL domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable Vlambda6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic Vlambda6 protein (Wil) that had neutral amino acids at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of immunoglobulin light chains.
Amino Acid Sequence↗
Derivation of 3D coordinate templates for searching structural databases: application to Ser-His-Asp catalytic triads in the serine proteinases and lipases.
It is well established that sequence templates (e.g., PROSITE) and databases are powerful tools for identifying biological function and tertiary structure for an unknown protein sequence. Here we describe a method for automatically deriving 3D templates from the protein structures deposited in the Brookhaven Protein Data Bank. As an example, we describe a template derived for the Ser-His-Asp catalytic triad found in the serine proteases and triacylglycerol lipases. We find that the resultant template provides a highly selective tool for automatically differentiating between catalytic and noncatalytic Ser-His-Asp associations. When applied to nonproteolytic proteins, the template picks out two "non-esterase" catalytic triads that may be of biological relevance. This suggests that the development of databases of 3D templates, such as those that currently exist for protein sequence templates, will help identify the functions of new protein structures as they are determined and pinpoint their functionally important regions.
Amino Acid Isomerases↗
Coverage of whole proteome by structural genomics observed through protein homology modeling database.
We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE ( http://daisy.nagahama-i-bio.ac.jp/Famsbase/ ), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics.
Amino Acid Sequence↗
The principles of enzyme stabilization. IV. Modification of 'key' functional groups in the tertiary structure of proteins.
The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.
Acetylation↗
Study of the tertiary structure of protein L11 from Escherichia coli ribosomes in solution by proton magnetic resonance.
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Bacterial Proteins↗
Analysis of the tertiary structure of protein beta-sheet sandwiches.
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Bence Jones Protein↗
Contribution of the free energy of mixing of hydrophobic side chains to the stability of the tertiary structure of proteins.
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Amino Acid Sequence↗
Correspondence of homologies in amino acid sequence and tertiary structure of protein molecules.
According to the method developed previously (Kubota, Y., Takahashi, S., Nishikawa, K. and Ooi, T. (1981) J. Theor, Biol. 91, 347-361), homology among proteins may be estimated quantitatively. We extended the method to investigate the relationship of an amino acid sequence to its teritary structure and identify homologous segments which have homologous native conformations in proteins. First, we selected proper indices for the computation of correlation coefficients from 32 properties inherent to amino acids, such as hydrophobicity. The arithmetic average of correlation coefficients using six indices gave rise to a good correlation for the CD- and EF-hand regions (Ca2+ binding sites) in carp parvalbumin, but poor ones for other segments. We then applied the method to homologous proteins, the three-dimensional structures of which are known: horse hemoglobin alpha-chain and beta-chain; cytochrome c and c2; serine proteases, chymotrypsinogen and elastase; alpha-lytic protease and protease A from prokaryotic organisms. The results show that the sequence homology estimated by the present method has a good correspondence to the homology in three-dimensional structures and therefore the method is promising for the identification of important sites in sequences which have similar native conformations. For an example of the application of the method, two sequences of human interferon, one from fibroblast and the other from leukocyte, are compared, suggesting functional sites in the molecule.
Amino Acid Sequence↗
Multiple molecular recognition mechanisms. Cytochrome P450--a case study.
Biomolecular recognition is complex. The balance between the different molecular properties that contribute to molecular recognition, such as shape, electrostatics, dynamics and entropy, varies from case to case. This, along with the extent of experimental characterization, influences the choice of appropriate computational approaches to study biomolecular interactions. Here, we present computational studies of cytochrome P450 enzymes and their interactions with small molecules and with other proteins. These interactions exemplify some of the diversity of molecular determinants of binding affinity and specificity observed for proteins and we discuss some of the challenges that they pose for molecular modelling and simulation.
Binding Sites↗
Armadillo: domain boundary prediction by amino acid composition.
The identification and annotation of protein domains provides a critical step in the accurate determination of molecular function. Both computational and experimental methods of protein structure determination may be deterred by large multi-domain proteins or flexible linker regions. Knowledge of domains and their boundaries may reduce the experimental cost of protein structure determination by allowing researchers to work on a set of smaller and possibly more successful alternatives. Current domain prediction methods often rely on sequence similarity to conserved domains and as such are poorly suited to detect domain structure in poorly conserved or orphan proteins. We present here a simple computational method to identify protein domain linkers and their boundaries from sequence information alone. Our domain predictor, Armadillo (http://armadillo.blueprint.org), uses any amino acid index to convert a protein sequence to a smoothed numeric profile from which domains and domain boundaries may be predicted. We derived an amino acid index called the domain linker propensity index (DLI) from the amino acid composition of domain linkers using a non-redundant structure dataset. The index indicates that Pro and Gly show a propensity for linker residues while small hydrophobic residues do not. Armadillo predicts domain linker boundaries from Z-score distributions and obtains 35% sensitivity with DLI in a two-domain, single-linker dataset (within +/-20 residues from linker). The combination of DLI and an entropy-based amino acid index increases the overall Armadillo sensitivity to 56% for two domain proteins. Moreover, Armadillo achieves 37% sensitivity for multi-domain proteins, surpassing most other prediction methods. Armadillo provides a simple, but effective method by which prediction of domain boundaries can be obtained with reasonable sensitivity. Armadillo should prove to be a valuable tool for rapidly delineating protein domains in poorly conserved proteins or those with no sequence neighbors. As a first-line predictor, domain meta-predictors could yield improved results with Armadillo predictions.
Amino Acid Sequence↗
Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus.
The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.
Binding Sites↗
Exploiting elements of transcriptional machinery to enhance protein stability.
The correlation between protein structure and function is well established, yet the role stability/flexibility plays in protein function is being explored. Here, we describe an in vivo screen in which the thermal stability of a test protein is correlated directly to the transcriptional regulation of a reporter gene. The screen readout is independent of the function of the test protein, proteolytic resistance, solubility or propensity to aggregate indiscriminately, and is thus dependent solely on the overall stability of the test protein. The system entails the use of an engineered chimeric construct that consists of three covalently linked domains; a constant N-terminal DNA-binding domain, a variable central test protein, and a constant C-terminal transcriptional activation domain. The test proteins are mutant variants of the beta1 domain of streptococcal protein G that span fairly evenly a thermal stability range from as low as 38 degrees C to above 100 degrees C. When the chimeric construct contains a test variant of low thermal stability, the reporter gene is up-regulated to a greater extent relative to that of more stable/less flexible variants. A panel of nine Gbeta1 mutant variants was used to benchmark the screen, and spectroscopic methods were employed to characterize the thermal and structural properties of each variant accurately. The screen was combined with in silico methods to interrogate a library of randomized variants for selection of mutants of greater structural integrity.
Amino Acid Sequence↗
Efficient determination of low-frequency normal modes of large protein structures by cluster-NMA.
The structure-function relationship is critical to understanding the biologically relevant functions of protein structures. Various experimental techniques and numerical modeling methods, normal mode analysis (NMA) in particular, have been employed to gain insight into this relationship. Experimental methods are often unable to provide all the desired information and comprehensive modeling techniques are often too computationally expensive. The authors build upon and optimize their cluster normal mode analysis (cNMA) tool, which uses embedded rigid-bodies and harmonic potentials to capture the biologically significant, low-frequency, oscillations of protein structures. cNMA represents atomic details with a scalable number of degrees-of-freedom, which can be chosen independent of structure size. This representation overcomes the otherwise quadratic order memory requirements and cubic order computational complexity associated with traditional all-atom NMA. cNMA is two orders of magnitude faster than traditional all-atom NMA when clustering by residue (very high resolution) and in the more traditional application using a fixed number of clusters, cNMA computationally scales as O(n), which is two orders of complexity faster than all-atom NMA. cNMA is presented and very large example structures with up to 10(6) atoms are analyzed on a notebook PC in the time scale of minutes/hours. The resulting mode shapes help identify biologically significant, conformational pathways.
Cluster Analysis↗
Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer.
Nrf2 regulates the cellular oxidative stress response, whereas Keap1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the Keap1 and Nrf2 interaction, we resolved the six-bladed beta propeller crystal structure of the Kelch/DGR and CTR domains of mouse Keap1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of Keap1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the beta propeller of Keap1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce Keap1's affinity for Nrf2. These results provide a structural basis for the loss of Keap1 function and gain of Nrf2 function.
Adaptor Proteins, Signal Transducing↗
Crustacean peptide and peptide-like pheromones and kairomones.
Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones are reviewed from a historical perspective. These crustacean information molecules are secondary functions of structural polymers. They are partial hydrolysis products, generated usually by the action of trypsin-like enzymes on proteins, and glycosidase enzymes on glycoproteins and proteoglycans. Structure-function studies based upon synthetic mimics of peptide information molecules show neutral amino acids with a basic carboxyl terminal are active in modifying physiological and or behavioral responses. Behaviorally active substituted amino sugar mimics are disaccharide hydrolysis products of heparin and chondroitin sulfate. Similar molecules are also used as information molecules by a variety of other marine organisms indicating they are a common biological theme.
Animals↗
Protein-water-ion interactions in a model of the pore domain of a potassium channel: a simulation study.
A model of the selectivity filter of a voltage-gated K+ (Kv) channel formed by an eight-stranded beta-barrel is compared with physiological properties of the channel. Continuum electrostatic calculations suggest that only two of the eight Asp sidechains at the extracellular mouth of the pore will ionise. A ring of four Tyr sidechains forms the narrowest region of the pore. Molecular dynamic simulations of the potential energy of a K+ ion as translated along the model pore indicate that the two ionised Asp sidechains and the hydroxyl groups of the Tyr sidechains stabilise the partially desolvated ion as it passes through the narrowest region.
Amino Acid Sequence↗