The molecular structure of Saccharomycopsis fibuligera alpha-amylase was predicted by a homology-based modeling technique, and the amino acid residues composing the active site were displayed with color codes according to their order of conservation. We noticed two highly conserved aromatic residues located in the active center, tyrosine 83 (Y83) and tryptophan 84 (W84), and examined their roles in catalytic activity by site-directed mutagenesis. The W, leucine (L), and asparagine (N) mutants at Y83 and the L mutant at W84 showed remarkable enhancement of transglycosylation activity and complementary decreases in native hydrolysis activity. The phenylalanine (F) mutant at Y83 and the F and Y mutants at W84 only decreased hydrolysis activity. Mechanistic and kinetic studies of these mutants using a reducing-end-blocked substrate and a hydrolysis-specific substrate revealed a probable transglycosylation mechanism and critical contributions of the 83rd and 84th aromatic residues to efficient hydrolysis. Given that aromatic residues stack against the faces of sugars, we assumed that Y83 and, presumably, W84 play roles in the binding of oligosaccharide substrates through the stacking interaction and in the indirect fixation of the catalytic water molecule through hydrogen bonding with the hydroxyl of the bound substrates. Mutations to nonaromatic residues could cause slight changes in the binding topology of substrates to favor transglycosylation over hydrolysis.

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Class I major histocompatibility complex (MHC) binds many short antigen peptides (9-11 amino acids) with diverse sequences derived from cytosolically degraded proteins, as part of the cytolytic immune response to defend the body against viral infection.1-4) To bind all these peptides, class I MHC primarily interacts with the N-and C-termini. 5,6) Additionally, the peptides bound with high affinity typically have conserved amino acid side chains, termed anchors, that bind in specific pockets primarily composed of polymorphic residues in a peptide-binding groove of class I MHC. 7-11)The combination of amino acids that bind at the anchor position is known as the peptide-binding motif.12-17) However, the above motifs are not sufficient to design a suitable peptide for binding because the binding affinity probably depends on sequence diversity.We are interested in the dynamics of the peptide-binding groove of class I MHC. To analyze the conformational dynamics of a protein, two standard approaches are used: molecular dynamic calculations and normal mode analysis. The former is free from harmonic approximation, but is only suitable for events in a time frame of 100 ps or faster. Moreover, the larger the molecule, the more the time frame is limited. Thus, to describe the dynamics of large biomolecules, normal mode analysis has been adopted, although a harmonic surface potential is used. [18][19][20][21][22][23][24][25] In this study, to describe the dynamic characteristics of class I MHC, we performed a normal mode analysis on HLA-A2 MHC that binds three peptides with different affinity: RT (ILKEPVHGV) is derived from HIV reverse transcriptase (residue 309-317).26) GP2 (IISAVVGIL) is derived from HER-2/neu (residue 654-662), and has a binding affinity weaker than that of RT despite the presence of peptide-binding motifs.27) I1Y (YLKEPVHGV) has a Tyr substituted for the amino-terminal end (Ile) of RT, and higher affinity than RT. 28,29) A normal mode analysis of HLA-A2 MHC complexed with the peptide (the complexed structure) and MHC alone (the peptide-removed structure) was performed. In both structures, fluctuations and motional correlation coefficients were calculated, and the differences compared among peptide-MHC complexes. ResultsFluctuation Changes in the Peptide-Binding Groove by Removing Antigen Peptides HLA-A2 MHC has two long a-helices (a1-helix group: residues 49-84, and a2-helix group: 140-179), which form a peptide-binding groove. The fluctuations of Ca atoms in the peptide-binding groove were compared among peptide-MHC complexes (Fig. 1). In all complexes, the profiles of fluctuations in all frequency modes were very similar to those in low frequency modes below 50 cm Ϫ1 (data not shown). The number of low frequency modes below 50 cm Ϫ1 ranged from 126 to 139 for both structures, and the ratio to total modes was ca. 7.5-8.5%.First, fluctuation changes in a1-helix group by removing antigen peptides were compared. In RT-MHC, 16 residues (residue 57-63, 65-71, 73-74) showed an increase in fluctuations, at aro...

The dynamics of crystal water molecules of guanosine dihydrate are investigated in detail by molecular dynamics (MD) simulation. A 2 ns simulation is performed using a periodic boundary box composed of 4 x 5 x 8 crystallographic unit cells and using the particle-mesh Ewald method for calculation of electrostatic energy. The simulated average atomic positions and atomic displacement parameters are remarkably coincident with the experimental values determined by X-ray analysis, confirming the high accuracy of this simulation. The dynamics of crystal water are analyzed in terms of atomic displacement parameters, orientation vectors, order parameters, self-correlation functions of the orientation vectors, time profiles of hydrogen-bonding probability, and translocations. The simulation clarifies that the average structure is composed of various stable and transient structures of the molecules. The simulated guanosine crystal forms a layered structure, with four water sites per asymmetric unit, classified as either interlayer water or intralayer water. From a detailed analysis of the translocations of water molecules in the simulation, columns of intralayer water molecules along the c axis appear to represent a pathway for hydration and dehydration by a kind of molecular valve mechanism.

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