Core I97L mutation in conjunction with P79Q is associated with persistent low HBV DNA and HBs antigen clearance in patients with chronic hepatitis B.

OBJECTIVES: When considering treatment for chronic hepatitis B (CHB), it is important to discriminate between patients with persistent low HBV DNA and patients with active hepatitis, who may proceed to cirrhosis. In this study, we sought to identify mutations in patients expected to have persistent low HBV DNA and ultimately exhibit clearance of hepatitis B surface antigen (HBsAg). METHODS: Serum samples were obtained from 33 CHB genotype C patients, divided based on HBV DNA and alanine aminotransferase (ALT) levels following observation for >2 years: Group A (n=10), transient HBV DNA ≥5.0 log copies/mL and ALT ≥120 IU/L; Group B (n=11), persistent HBV DNA <5.0 and ALT <60; and Group C (n=12), persistent HBV DNA <4.0 and ALT <30. Full-length HBV sequences were compared among groups. Subsequently, 82 patients with CHB were evaluated for the I97L mutation and the additional mutation P79Q. We compared cumulative incidences of persistent low HBV DNA and HBsAg clearance in patients with or without I97L and P79Q by the Kaplan-Meier method. RESULTS: Incidence of Core mutation I97L differed significantly among groups: A, 30% (3/10); B, 36.4% (4/11); C, 83.3% (10/12) (p = 0.021). Cumulative incidences of persistent low HBV DNA and HBsAg clearance were significantly higher in patients with I97L than in those with wild-type I97 (p = 0.003 and p = 0.016, respectively), and even higher in those with P79Q. CONCLUSIONS: In patients with CHB, measurement of I97L and additional mutation P79Q would be useful for predicting persistent low HBV DNA, normal ALT, and HBsAg clearance.

Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

An automatic homology modeling method consisting of database searches and simulated annealing.

We introduce a method of homology modeling consisting of database searches and simulated annealing. All processes involving searches for homologous proteins, alignment, the construction of Calpha atoms, construction of main-chain atoms, and the construction of side-chain atoms are performed automatically. In this method, main-chain conformations are generated from the weighted average of mainchain coordinates in reference proteins. The weight is defined by the local space homology representing the similarity of environmental residues at topologically equivalent positions in reference proteins. Side-chain conformations are generated for constructed main-chain atoms by database searches, and main-chain atoms are optimized for the fixed side-chain conformations. These two processes, i.e., the side-chain generation and main-chain optimization, are repeated several times. This type of construction provides a structure similar to the x-ray structure, in particular, for main-chain and side-chain atoms in the residues belonging to structurally conserved regions (SCRs). The accuracy of our method was evaluated for 14 proteins whose structures are known. The average root mean square deviation between models and x-ray structures was 2.29 A for all atoms, and the percentage of chi1 angles within 30 degrees was 72.6% for SCRs residues. Some models were in good agreement with their respective x-ray structures. Our method, which has the advantage of being automated, gives results similar to, or better than, published results for three widely used test proteins. Our software, FAMS, is available on the World Wide Web.

Intermediate state during the crystal transition in aspartame, studied with thermal analysis, solid-state NMR, and molecular dynamics simulation.

Aspartame (L-alpha-aspartyl-L-phenylalanine methyl ester) is a dipeptide sweetener about 200 times as sweet as sugar. It exists in crystal forms such as IA, IB, IIA, and IIB, which differ in crystal structure and in the degree of hydration. Among these, IIA is the most stable crystal form, and its crystal structure has been well determined (Hatada et al., J. Am. Chem. Soc., 107, 4279-4282 (1985)). To elucidate the structural factors of thermal stability in the IIA form of aspartame and to examine the physical process in the crystal transformation between the IIA and IIB forms, we performed a thermal analysis and solid-state NMR measurements. We found that a quasi-stable intermediate state exists in the transformation, and it has the same crystal lattice as the usual IIA form, despite the dehydration from 1/2 mol to 1/3 mol per 1 mol of aspartame. The results of the energy component analysis and the molecular dynamics simulation suggest that the entropic effect promotes the generation of the intermediate state, which is presumably caused by the evaporation of the water of crystallization and the increase of molecular motion in aspartame. Thus, the thermal stability of the IIA form is attributable to a structural property, i.e., the crystal lattice itself is retained during the above dehydration. Moreover, the molecular dynamics simulations suggest that the aspartame molecules have two kinds of conformational flexibility in the intermediate state.

Dynamic character of the complex of human blood coagulation factor VIIa with the extracellular domain of human tissue factor: a normal mode analysis.

As an attempt to investigate the dynamic interactions between plasma serine protease, coagulation factor VIIa (VIIa) and its cofactor, tissue factor (TF), we performed normal mode analysis (NMA) of the complex of VIIa with soluble TF (the extracellular part of TF; sTF). We compared fluctuations of Calpha atoms of VIIa or sTF derived from NMA in the VIIa-sTF complex with those of VIIa or sTF in an uncomplexed condition. The atomic fluctuations of the Calpha atoms of sTF complexed with VIIa did not significantly differ from those of sTF without VIIa. In contrast, the atomic fluctuations of VIIa complexed with sTF were much smaller than those of VIIa without sTF. These results suggest that domain motions of VIIa molecule alone are markedly dampened in the VIIa-sTF complex and that the sTF molecule is relatively more rigid than the VIIa molecule. This may indicate functions of TF as a cofactor.

Structural studies of the interactions of normal and abnormal human plasmins with bovine basic pancreatic trypsin inhibitor.

Catalytic activity of human plasmin is inhibited by bovine basic pancreatic trypsin inhibitor (BPTI, also known as aprotinin). In spite of increased interest in the function of BPTI as an inhibitor of plasmin, the 3-D structure of the plasmin-BPTI complex has not yet been determined. Therefore, in the present paper, the structure of the plasmin-BPTI complex was constructed by the homology modeling method, which provided information about the high affinity of plasmin for BPTI. Moreover, normal mode analyses of free plasmin, free BPTI and the plasmin-BPTI complex were carried out to investigate the changes in dynamics following complex formation. After study of the plasmin-BPTI interaction, we also investigated the binding of BPTI with abnormal plasmin, theoretically and experimentally. The result showing that BPTI binds to abnormal plasmin in the same way as it does to normal plasmin supports the previous finding that the difference between normal and abnormal plasmins is very small and that the abnormality is localized to the catalytic site.

A homology modeling method of an icosahedral viral capsid: inclusion of surrounding protein structures.

A methodological development is presented for homology modeling of an icosahedrally symmetric assembly of proteins. In the method, a main-chain structure of an asymmetric unit of a protein assembly is constructed and structure refinement is performed, taking the surrounding symmetry-related proteins into consideration with rotational symmetry boundary conditions. To test the procedure, three models of a poliovirus capsid were constructed with different modeling conditions based on the X-ray structure of a rhinovirus capsid. Model S and model N were constructed with and without considering surrounding proteins, respectively. Model N2 was obtained by refinement in rotational symmetry boundary conditions of the structure of model N. The three models were compared with the X-ray structure of a poliovirus capsid. Root mean square deviations and C alpha distances indicate that model S is the most accurate. Examination of the intermolecular short contacts indicates that model S and model N2 are superior to model N, because they do not make severe intermolecular short contacts. Symmetric intermolecular interactions are important for both the structural fragment search and energy minimization to predict better loop structures. The programs developed in this study are thus valuable in homology modeling of an icosahedral viral capsid.

Effect of exceptional valine replacement for highly conserved alanine-55 on the catalytic site structure of chymotrypsin-like serine protease.

The catalytic triad consisting of His57, Asp102 and Ser195, which is completely conserved within the chymotrypsin-like serine protease family, plays a central role in catalysis. Highly conserved Ala55 also likely plays an important role in catalysis due to its location just behind the catalytic triad. The only exception to the conserved Ala55 in mammalian serine proteases is Val55 in bovine protein C. Interestingly, it has been demonstrated that the replacement of Ala55 with Thr results in the reduced activity of plasmin in patients with venous thrombosis and with retinochoroidal vascular disorders, which indicates the importance of Ala55 in catalysis. In the present study, we constructed a bovine protein C model which shows that Val55 causes no serious rearrangement of the catalytic site structure. We also constructed an A55T variant model of trypsin for comparison. The A55T substitution alters His57 into an inactive conformation, forming an unusual hydrogen bond between Thr55 O gamma 1 and His57 N epsilon 2. The present study shows that the Ala/Val55 residue contributes heavily to the active conformation of His57 and enables His57 to accept a proton from Ser195 O gamma effectively.

Dynamic structures of granulocyte colony-stimulating factor proteins studied by normal mode analysis: two domain-type motions in low frequency modes.

In this study, granulocyte colony-stimulating factor (GCSF) proteins were chosen as subjects for normal mode analysis. As helical cytokines with a four helix bundled type topology, they were classified into long chain and short chain groups by Sprang and Bazan. Normal mode calculations were also carried out with leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and growth hormone (GH) as members of the long chain group and GCSF and IL-2 and IL-4 as members of the short chain group. For the GCSF families it was found that the fluctuations in the helical region are smaller than in the loop region, and it is clear that on the whole the smaller fluctuation residues belong to a large hydrophobic core region. Thus, it can be imagined how the receptor binding sites approach the receptor within the normal time-scale of pico seconds. In addition, two similar domain-type motions in low frequency modes were found with proteins in the long chain group, although we never observed any sequence similarity in the two separate two-domain regions in each protein of the long chain group. On the other hand, these two domain-type motions were not clear in proteins of the short chain group.

The role played by environmental residues on sidechain torsional angles within homologous families of proteins: a new method of sidechain modeling.

We investigated the conservation of sidechain conformation for each residue within a homologous family of proteins in the Protein Data Bank (PDB) and performed sidechain modeling using this information. The information was represented by the probability of conserved sidechain torsional angles obtained from many families of proteins, and these were calculated for a pair of residues at topologically equivalent positions as a result of structural alignment. Probabilities were obtained for a pair of same amino acids and for a pair of different amino acids. The correlation between environmental residues and the fluctuation of probability was examined for the pair of same amino acid residues, and the simple probability was calculated for the pair of different amino acids. From the results on the same amino acid pairs, 17 amino acids, except for Ala, Gly, and Pro, were divided into two types: those that were influenced and those that were not influenced by the environmental residues. From results on different amino acid pairs, a replacement between large residues, such as Trp, Phe, and Tyr, was performed assuming conservation of their torsional angles within a homologous family of proteins. We performed sidechain modeling for 11 known proteins from their native and modeled backbones, respectively. With the native backbones, the percentage of the chi 1 angle correct within 30 degrees was found to be 67% and 80% for all and core residues, respectively. With the modeled backbones, the percentage of the correct chi 1 angle was found to be 60% and 72% for all and core residues, respectively. To estimate an upper limit on the accuracy for predicting sidechain conformations, we investigated the probability of conserved sidechain torsional angles for highly similar proteins having > 90% sequence identity and < 2.5-A X-ray resolution. In those proteins, 83% of the sidechain conformations were conserved for the chi 1 angle.

The carboxyl-terminal region of protein C is essential for its secretion.

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

Arg260-Cys mutation in severe factor XIII deficiency: conformational change of the A subunit is predicted by molecular modelling and mechanics.

To explore the implications of the structure/ function relationships in factor XIII. a patient with severe A subunit deficiency was examined at the DNA and RNA levels. Nucleotide sequence analysis of the patient's DNA amplified by PCR revealed that the patient had a replacement of C by T in the codon for Arg260. RT-PCR analysis demonstrated that only one kind of mRNA coding for the Arg260-Cys mutation was expressed in the patient at a normal level. Another possible defective allele of the A subunit gene with a G-A polymorphism was not expressed (null allele). The substitution of Arg260 by Cys located on the interface of two A subunits would preclude the reciprocal ionic interaction (salt bridge) between Arg260 and Asp404. Molecular modelling and, for the first time, molecular mechanics calculated that Cys260 changed the local conformation of the A subunit and reduced the electrostatic interaction between two monomers, suggesting destabilization of the molecule's dimer.

Elucidation of the cause for reduced activity of abnormal human plasmin containing an Ala55-Thr mutation: importance of highly conserved Ala55 in serine proteases.

In serine proteases, Ala55 is highly conserved and located just behind the catalytic triad. That the activity of human plasmin is reduced by the A55T substitution indicates the importance of Ala55 in catalysis. In the present study, the 3-D model of A55T human plasmin shows that an unusual hydrogen bond between Thr55 Ogamma1 and His57 Nepsilon2 alters His57 into an inactive conformation in which His57 cannot accept a proton from Ser195 as a catalytic base. Our results demonstrate that Ala55 contributes heavily to the active conformation of His57 and ensures the proton transfer from Ser195 to His57.

Molecular mechanisms of type II factor XIII deficiency: novel Gly562-Arg mutation and C-terminal truncation of the A subunit cause factor XIII deficiency as characterized in a mammalian expression system.

To explore the biological and clinical implications of the structure/function relationships in factor XIII, mutations in two patients with type II deficiency were identified and characterized in a mammalian expression system. Nucleotide sequence analysis of the A subunit gene showed that case no. 1 had a deletion of 4 bp (AATT) in exon XI and that, in case no. 2, Gly562 (GGG) had been replaced by Arg(AGG). The deletion in case no. 1 leads to a premature termination at codon 464. Restriction digestion of amplified DNAs confirmed that both cases were homozygous for their respective mutations. Reverse transcription-polymerase chain reaction analysis demonstrated that the level of mRNA was greatly reduced in case no. 1, whereas the level of mutant mRNA expressed in case no. 2 was normal. Molecular modeling calculated that Arg562 changed the conformation of the A subunit, suggesting misfolding and/or destabilization of the molecule. To determine how these mutations impaired synthesis of the A subunit, recombinant A subunits bearing the mutations were expressed in mammalian cells. Pulse-chase experiments showed that the mutants were synthesized normally but disappeared rapidly, whereas the wild-type remained. These results indicate that both mutant proteins with an altered conformation become prone to rapid degradation, resulting in factor XIII deficiency in these patients.

Factor X Nagoya 1 and Nagoya 2: a CRM- factor X deficiency and a dysfunctional CRM+ factor X deficiency characterized by substitution of Arg306 by Cys and of Gly366 by Ser, respectively.

We have identified, in two unrelated patients, factor X deficiency that we have designated factor X Nagoya 1 and Nagoya 2, respectively. The proband with factor X Nagoya 1 showed factor X activity level of 3% and factor X antigen level < 10% of the normal control value. All the exons and intron/exon junctions of the factor X gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and nonradioactive single-strand conformational polymorphism (SSCP) analysis. Exon 8 containing DNA fragment of the proband with factor X Nagoya 1 showed aberrant migration on SSCP analysis. All exon-containing DNA fragments amplified by PCR were sequenced, and we identified a C-to-T substitution in exon 8 in the human factor X gene of the proband, which results in the replacement of Arg306 by Cys. This genetic defect has been transmitted from her father, and her sister also carried the same mutation; both showed almost half the normal levels of both factor X activity and antigen. The coordinates of human factor Xa indicated that Arg306 in the catalytic domain is positioned at the beginning of the alpha-helix near the second EFG-like domain. The substitution for Arg of Cys has been supposed to cause the destruction of local alpha-helix formation, possibly leading to the secretion problem. The proband with dysfunctional factor X Nagoya 2 was characterized by factor X activity level of 34% with normal factor X antigen level of 80%. We identified one substitution of G for A in exon 8 in the human factor X gene of the proband, which results in the replacement of Gly366 by Ser. As the Gly366 is positioned at the primary substrate binding pocket. the replacement of Gly with Ser would cause a defect of substrate binding, leading to the loss of enzymatic activity.

Amino acid similarity matrix for homology modeling derived from structural alignment and optimized by the Monte Carlo method.

In this paper, we obtained a similarity matrix for homology modeling based on the structure of proteins in a structural alignment. The alignment procedure was executed within dynamic programming generally used in alignment methods. An initial matrix derived from the structural alignment was optimized by the Markov chain Monte Carlo method at low temperature to fit its sequence alignment to the structural alignment. Structural alignment was performed on the basis of the superposition of C alpha atoms for two protein structures. The objective function in the Monte Carlo procedure was defined by entropy in the information theory, allowing us to show that the amino acid similarity matrix aligned accurately. When compared with the structural alignment, the average number of incorrect amino acid residues in the sequence alignment was 22.6 for all residues and about 3.7 for residues in structurally conserved regions. The alignment with our matrix was more similar to structural alignment than to sequence alignments using other amino acid substitution matrices.

A computer modeling study of the interaction between tissue factor pathway inhibitor and blood coagulation factor Xa.

Activation of blood coagulation factor X to factor Xa (FXa) is inhibited by tissue factor pathway inhibitor (TFPI). The second Kunitz-type inhibitory domain (K2) of TFPI binds a catalytic domain of FXa, whereas the first domain (K1) does not. We analyzed computer models of complexes of FXa with K1 or K2, which were made using a crystal structure of FXa. Favorable hydrophobic interaction was observed in the complex of FXa with K2. Furthermore, we constructed a tertiary structure of FXa using CHIMERA to assess the accuracy of a homology modeling method. The isolated model structure of FXa agreed well with the crystal structure, but analyses of complexes of this structure with K1 or K2 revealed that the models of complexes could not provide clear evidence of greater binding ability to K2 because of the positional difference of a few side chains interacting with the inhibitor.

A theoretical approach to the bell-shaped dependency of cell proliferation on the hormone concentration.

Binding of human growth hormones (hGH) to the receptors was studied with a theoretical, molecular-level model. An hGH has two sites bindable to different receptors with different binding energies. In the model the hGHs diffusively moved in a box (i.e. the volume of solution), and receptors on the bottom face the box (i.e. a membrane). The system consisted of a number of hGHs and receptors, which could form hGH-[receptor]2 or hGH-receptor complexus. In a complex, small inter-molecular positional fluctuations were allowed with keeping the inter-molecular binding. Partition function of the system was calculated. In a low hGH-concentration range, free receptors were dominant on the membrane; in a medium concentration range, hGH-[receptor]2 complexus, which induce cell-proliferation, were dominant, and in a high concentration range, hGH-receptor complexus, which inhibit the proliferation, were dominant. This dependency (bell-shaped dependency) of formation of hGH-[receptor]2 complex on the hGH concentration agreed well with experimental observation. The values of EC50 (hGH concentration at that the cell-proliferation rate rose to 50% of the maximum rate by the formation of hGH-[receptor]2 complexus) and IC50 (hGH concentration at that the proliferation rate decreased to 50% of the maximum by the formation of hGH-receptor complexus) from my method were 18 pM and 2.2 microM, respectively. By calculating thermodynamic quantities (i.e. entropy and enthalpy), factors that determine the bell-shaped dependency were obtained. At the medium concentration, the entropy of free hGHs played an important role in stabilizing the hGh-[receptor]2 complex. Small changes in binding energies or in inter-molecular positional fluctuations largely changed the dependency of the complex formation on the hGH concentration. This method is useful in explaining the experimental results that small molecular modification largely changes the formation of the complex.

Prediction of protein side-chain conformations by principal component analysis for fixed main-chain atoms.

A method of side-chain prediction without calculating the potential function is introduced. It is based on the assumption that similar side-chain conformations have a similar structural environment around the side chains. The environment information is represented by vectors that were obtained from principle component analysis and represented by the variance of positions of main-chain atoms around side chains. This information was added to the side-chain library (rotamer library) made from X-ray structures. Side-chain conformations were constructed using this side-chain library without using potential functions. An optimal solution was determined by comparing environmental information with the backbone conformation around the side chain to be predicted and native ones in the library. The method was performed for 15 proteins whose structures were known. The result for the root-mean-square deviation between the predicted and X-ray side-chain conformations was approximately 1.5 A (the value for core residues was approximately 1.1 A) and the percentage of predicted chi 1 angles correct within 40 degrees was approximately 65% (75% for the core). The computational time was short (approximately 60 s for the prediction of proteins with 200 amino acid residues). About 70% of the side-chain conformations were constructed by location of the main-chain atoms around the central C beta atom and the average of r.m.s.d. was approximately 1.4 A (for core residues the average was approximately 1.0 A).