Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.     

A novel strategy for defining critical amino acid residues involved in protein/glycosaminoglycan interactions

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.     

Identification and functions of amino acid residues in PotB and PotC involved in spermidine uptake activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.     

Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

Background  

Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.     

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV.

We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.     

Defining the LGR8 residues involved in binding insulin-like peptide 3

The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [(125)I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.     

Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck delta II crystallin

Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity. At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate. The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis. On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined. Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity. Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities. During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved [Vallee et al. (1999) Biochemistry 38, 2425-2434], which has allowed the kinetic data to be interpreted on a structural basis.     

Identification of thromboxane synthase amino acid residues involved in heme-propionate binding

Thromboxane A2 synthase (TXAS) is a member of the cytochrome P450 superfamily and catalyzes an isomerization reaction that converts prostaglandin H2 to thromboxane A2. As a step toward understanding the structure/function relationships of TXAS, we mutated amino acid residues predicted to bind the propionate groups of A- and D-pyrrole rings of the heme. These mutations at each of these residues (Asn-110, Trp-133, Arg-137, Arg-413, and Arg-478) resulted in altered heme binding, as evidenced by perturbation of the absorption spectra and EPR. The mutations, although causing no significant changes in the secondary structure of the proteins, induced tertiary structural changes that led to increased susceptibility to trypsin digestion and alteration of the intrinsic protein fluorescence. Moreover, these mutant proteins lost their binding affinity to the substrate analog, had a lower heme content and retained less than 5% of the wild-type catalytic activity. However, mutations at the neighboring amino acid of the aforementioned residues yielded mutant proteins retaining the biochemical and biophysical properties of the wild type TXAS. Aligning the TXAS sequence with the structurally known P450s, we proposed that in TXAS the A-ring propionate of the heme is hydrogen bonded to Asn-110, Arg-413, and Arg-478, whereas D-ring propionate is hydrogen bonded to Trp-133 and Arg-137. Furthermore, both A- and D-ring propionates bulge away from the heme plane and both lie on the proximal face of heme plane, a structure similar to P450terp.     

Identification and mutational analysis of amino acid residues involved in dipyridamole interactions with human and Caenorhabditis elegans equilibrative nucleoside transporters

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.     

Conserved amino acid networks involved in antibody variable domain interactions

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody V_H and V_L function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which V_H and V_L domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (?‐values). We provide rosters of the key conserved amino acid pairs in antibody V_H and V_L domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in V_H and V_L are at or adjacent to the V_H–V_L interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the V_H domain, but not the V_L domain, residue pairs at the variable‐constant domain interface (V_H–C_H1 interface) are also strongly conserved. The same network of conserved V_H positions involved in interactions with both the V_L and C_H1 domains is found in camelid V_HH domains, which have evolved to lack interactions with V_L and C_H1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

The nature of intermolecular interactions between aromatic amino acid residues

The nature of intermolecular interactions between aromatic amino acid residues has been investigated by a combination of molecular dynamics and ab initio methods. The potential energy surface of various interacting pairs, including tryptophan, phenilalanine, and tyrosine, was scanned for determining all the relevant local minima by a combined molecular dynamics and conjugate gradient methodology with the AMBER force field. For each of these minima, single-point correlated ab initio calculations of the binding energy were performed. The agreement between empirical force field and ab initio binding energies of the minimum energy structures is excellent. Aromatic-aromatic interactions can be rationalized on the basis of electrostatic and van der Waals interactions, whereas charge transfer or polarization phenomena are small for all intermolecular complexes and, particularly, for stacked structures. Proteins 2002;48:117-125.     

Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.     

Classification of amino acids based on comparative analysis of contacts in DNA-protein complexes and specific DNA-protein interactions

We propose a classification of amino acid residues based on the events of contact formation between particular residues and DNA nucleotides, i.e., using the most integral properties that characterize interactions organizing DNA-protein complexes. We apply the Voronoi-Delaunay tessellation to draw statistics of contacts and of contact areas for a set of 1937 DNA-protein complexes. Similarity of amino acid residues is defined upon comparison of corresponding rows and matrices of contacts and areas of contacts. Nine measures of distance have been used to estimate the closeness of rows. Residues have been grouped by three hierarchical and two nonhierarchical clustering methods. In a total tree built using nine metrics with three hierarchical methods, we show that clustering centers (pairs of amino acids) in the main groups are always constant while other relationships between objects vary. Major classes of up to six amino acids correspond to certain local structures of the polypeptide chain. These data can be taken into account when designing DNA-protein ligands.     

Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human 5-lipoxygenase.

Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5-lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron.     

Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs

We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.     

Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III

Background

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca^2 +-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores.

Methods

Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined.

Results

K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane.

Conclusions

Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III.

General significance

The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.     

Synthesis and activity of dermorphin analogues containing unusual amino acid residues

Summary Solid phase syntheses of analogues of the opioid heptapeptide dermorphin (H-Tyr-dAla-Phe-Gly-Tyr-Pro-Ser-NH₂) containing in the first position 3-aminotyrosine, 3-nitrotyrosine, 4-aminophenylalanine, or nucleoamino acids, 3-(uracilyl-1)alanine, 3-(thyminyl-1)alanine and 3-(6-methyluracilyl-1)alanine are described. The receptor binding properties and analgesic activity of the analogues were examined in comparison with dermorphin. All analogues showed low opioid activity in the binding assays with respect to μ- and δ-receptors. The peptide containing 3-(thyminyl-1)alanine demonstrated a high analgesic activity in different tests when administered intracisternally in mice.