Contribution Of Amino Acid Residues Research Articles

Identification of Amino Acid Residues in the Human Immunodeficiency Virus Type-1 Reverse Transcriptase Tryptophan-repeat Motif that are Required for Subunit Interaction Using Infectious Virions

The human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) functions as a heterodimer (p51/p66), which makes disruption of subunit interactions a possible target for antiviral drug design. Our understanding of subunit interface interactions has been limited by the lack of virus-based approaches for studying the heterodimer. Therefore, we developed a novel subunit-specific mutagenesis approach that enables precise molecular analysis of the heterodimer in the context of infectious HIV-1 particles. Here, we analyzed the contributions of amino acid residues comprising the Trp-motif to RT subunit interaction and function. Our results reveal important inter- and intra-subunit interactions of residues in the Trp-motif. A tryptophan cluster in p51 (W398, W402, W406, W414), proximal to the interface, was found to be important for p51/p66 interaction and stability. At the dimer interface, residues W401, Y405 and N363 in p51 and W410 in p66 mediate inter-subunit interactions. The W401 residue is critical for RT dimerization, exerting distinct effects in p51 and p66. Our analysis of the RT heterodimerization enhancing non-nucleoside RT inhibitor (NNRTI), efavirenz, indicates that the effects of drugs on RT dimer stability can be examined in human cells. Thus, we provide the first description of subunit-specific molecular interactions that affect RT heterodimer function and virus infection in vivo. Moreover, with heightened interest in novel RT inhibitors that affect dimerization, we demonstrate the ability to assess the effects of RT inhibitors on subunit interactions in a physiologically relevant context.

蛋白質立体構造から安定化のメカニズムを定量的に理解する方法

Mutant proteins are useful for study on the mechanism of protein stability. However, it is difficult to evaluate the changes in stability due to mutation, because contributions of amino acid residues to the conformational stability differ depending on their locations even if the same residues are substituted. In order to evaluate each contribution of several factors responsible for protein stability, we determined the crystal structures and measured the protein stability for more than a hundred of mutant human lysozymes. Analyzing this structure/stability database, the parameters of each stabilization factor, such as hydrophobic effect and hydrogen bond, could be estimated. These parameters are available for understanding the thermostabilization mechanism of the proteins from hyperthermophiles and for the computer-aided drug design.

Partition of protein solvation into group contributions from molecular dynamics simulations

Linear response theory coupled to molecular dynamics simulations with an explicit solvent representation is used to derive fractional contributions of amino acid residues to the solvation of proteins. The new fractional methods developed here are compared with standard approaches based on empirical 1D and 3D statistical potentials, as well as with estimates obtained from the analysis of classical molecular interaction potentials. The new fractional methods, which have a clear physical basis and explicitly account for the effects due to protein structure and flexibility, provide an accurate picture of the contribution to solvation of different regions of the protein.

Analysis of structural statistical properties of proteases and nonproteases

The different structural properties of proteases and nonproteases are well investigated in this paper. The average percentage P L of residues having a number of long-range contacts greater than or equal to N L(≥ N L) for the proteases is larger than that for the nonproteases. The average number of long-range contacts per residue P L,μ in four secondary structure μ ( μ=H, E, T, and N) for the proteases is also larger than that for the nonproteases. We calculate the average contact order (CO) per protein, the average long-range order (LRO) per protein, and the average total contact distance (TCD) per protein, and find that the average value of LRO for the nonproteases is smaller than that for the proteases. However, both proteases and nonproteases have the same average values of CO and TCD. The average number of long-range contacts per residue C L for the proteases is larger than that for the nonproteases, however, the average number of short-range contacts per residue C S for the proteases is smaller than that for the nonproteases. It is also shown that the square of radius of gyration for the proteases is relatively smaller than for the nonproteases. This finding implies that proteases are more compact than nonproteases. In protein molecule, each residue has a different ability to form contacts, and in general the number of residues having a small number of contacts is greater than that having a large number of contacts. Here we have concluded that the probability P( n) of amino acid residues having n pairs of contacts in all residues fits a good Gaussian distribution, and there has the same form of Gaussian distribution for 20 amino acid residues. The most probable number of contacts, n C, for the proteases is greater than that for the nonproteases for 20 amino acid residues and one has a good correlation with the Fauchere-Pliska hydrophobicity (FPH) scale. Finally we discuss the relative contribution of amino acid residues involved in cation–π interactions. The higher fraction of cation–π interactions observed in the proteases is found to be reflection of the more general, more frequent occurrence of these interactions in these proteins. All these findings would be helpful for us to understand structural differences between the proteases and other proteins.

A mutant phospholipase D with enhanced thermostability from Streptomyces sp.

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329–441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.

The role of the zinc finger motif and of the residues at the amino terminus in the function of yeast ribosomal protein YL37a

YL37a is an essential yeast ribosomal protein that has a C 2-C 2 zinc finger motif. Replacement of the cysteine residues had yielded variants that lacked the capacity to bind zinc but still supported cell growth. In a continuation of an examination of the relation of the structure of YL37a to its function, the contribution of amino acid residues in the intervening sequence between the internal cysteine residues of the motif was evaluated. Substitutions of alanine for the lysine residues at positions 44, 45, or 48, or for arginine 49 slowed cell growth. The most severe effect was caused by a double-mutation, K48A-R49A. A mutation of tryptophan 55 to alanine was lethal. Mutations to alanine of six conserved residues (K6, K7, K13, Y14, R17, and Y18) in the amino-terminal region decreased cell growth; the Y14 mutation was lethal. An in vitro assay for binding of YL37a to individual 26 S rRNA domains was developed. Binding of the recombinant fusion protein MBP-YL37a was to domains II and III; the K d for binding to domain II was 79 nM; for domain III it was 198 nM. There was a close correspondence between the effect of mutations in YL37a on cell growth and on binding to 26 S rRNA. In the atomic structure of the 50 S subunit of Haloarcula marismortui, the archaebacteria homolog of yeast YL37a, L37ae, coordinates a zinc atom and the finger motif is folded and interacts mainly with domain III of 23 S rRNA; whereas the amino-terminal region of L37ae interacts primarily with domain II. The biochemical and genetic experiments complement the three-dimensional structure and define for the first time the functional importance of a subset of the residues in close proximity to nucleotides.

How to Evaluate the Contribution of Factors to the Conformational Stability of a Protein.

Mutant proteins are useful for study of protein stability. However, it is difficult to explain the changes in stability due to mountain, because contributions of amino acid residues to the conformational stability are quite different, depending on their location. In order to quantify directly the contribution of several factors to the conformational stability of a protein, we determined the thermodynamic parameters of denaturation and the crystal structures for more than 60 mutant human lysozymes. Analyzing this database (stability/structure database), the contribution of some factors to conformational stability of a protein, such as hydrophobic effect, hydrogen bond, and water molecule, could be estimated.

A New Method to Characterize Hydrophobic Organization of Proteins: Application to Rational Protein Engineering of Barnase

We present a new algorithm for characterization of protein spatial structure basing on the molecular hydrophobicity potential approach. The method is illustrated by the analysis of three-dimensional structure of barnase and barnase-barstar complex. Current approach enables identification of amino acid residues situated in unfavorable environment (these residues may be “active” for binding), and to map quantitatively hydrophobic, hydrophilic and unfavorable hydrophobic-hydrophilic intra-and inter-molecular contacts involving backbone and side-chain segments of amino acid residues. Calculation of individual contributions of amino acid residues to such contacts permits identification of structurally-important residues. The contact plots obtained with molecular hydrophobicity potential calculations, provide easy rules to choose sites for mutations, which can increase a strength of intra- or inter-molecular hydrophobic interactions. The unfavorable hydrophobic-hydrophilic contact can be mutated to favorable hydrophobic, and already existing weak hydrophobic contact can be strengthen by increasing hydrophobicity of residues in contact. Basing on the analysis of the contact plots, we suggest several mutations of barnase which are supposed to increase intramolecular hydrophobic interactions, and thus might lead to increased stability of the protein. Part of these mutations was studied previously experimentally, and indeed stabilized barnase. The other of predicted mutations were not studied experimentally yet. Several new mutations of barnase and barstar are also proposed to enhance the hydrophobic interactions on their binding interface.

Tests for the fractional active-site model in Flp site-specific recombination. Assembly of a functional recombination complex in half-site and full-site strand transfer

The Arg191-His305-Arg308 (the RHR triad) and Tyr343 of Flp site-specific recombinase correspond to the invariant tetrad residues of the integrase family of proteins. Flp mutants altered at these positions are blocked at the strand cleavage or the strand exchange step of recombination. Hybrid half-site-recombinase complexes formed by step-arrest mutants of Flp have revealed that an Flp monomer occupying a half-site does not cleave that half-site but rather cleaves a half-site occupied by a second Flp monomer. This trans-DNA cleavage is neatly accommodated by a model in which an Flp active site is assembled by contribution of amino acid residues from at least two protein monomers. Using a combination of wild type Flp, single, double, and triple step-arrest Flp mutants, critical predictions of the fractional active-site model have been verified. First, a wild type monomer paired with an RHR triad-Tyr343 double mutant is a catalytically inactive combination. Second, each pairwise combination of a single, double, or triple RHR mutant with Flp (Y343F) yields approximately equivalent levels of catalytic complementation. Half-site to half-site and half-site to full-site crosses suggest that execution of a strand transfer event within a half-site and between a half-site and a full site requires dimeric and tetrameric Flp configurations, respectively.

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.

Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis.

Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive. In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions. For other oligomers, catalysis requires the contributions of amino acid residues at the interface between adjacent polypeptide chains, and monomers are inactive because they cannot form complete active sites. A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parental mutants produced by site-directed mutagenesis. This approach was applied in a study of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala. Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 10(5) more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites. Hybrids from the His and Lys mutants had comparable specific activities. Moreover, one hybrid with approximately 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer. As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84----Gln and His-134----Ala) trimers. The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers. This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains. The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamoylase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomeric enzymes.