Detection of subunit interfacial modifications by tracing the evolution of clamp–loader complex

Mihoko Saito1, Takuji Oyama2 and Tsuyoshi Shirai3,4

1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 and Departments of 2Structural Biology and 3Computational Biology, Biomolecular Engineering Research Institute, 6–2–3 Furuedai, Suita, Osaka 565-0874, Japan

4 To whom correspondence should be addressed. E-mail: shirai{at}beri.or.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The archaeal and eukaryal clamp–loader and clamp proteins were investigated with the evolutionary trace method. The molecular phylogeny of the proteins suggested that the hetero-pentameric complex of the archaeal clamp–loader with two subunits (RFCL and RFCS) was not a preserved ancestral type, but a degenerated version of the eukaryal complex of five subunits (RFC1-5). The evolutionary trace of amino acid replacements during the course of subunit differentiation revealed that the replacements had accumulated preferentially at the subunit interface regions. Some of the interfacial modifications that might be responsible for the specific interaction between the subunits were conserved in the current complex.

Keywords: evolutionary trace/PCNA/protein complex/protein interface/RFC


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Clamp–loader proteins catalyze the loading reaction of clamp protein on DNA at replication origins or DNA lesions (Tsurimoto and Stillman, 1990Go; Stillman, 1994Go; O'Donnell et al., 2001Go; Jeruzalmi et al., 2002Go). The clamp protein is a ring-shaped molecule that is topologically linked to a DNA molecule, so that the DNA polymerase attached to the clamp has increased processivity on the DNA molecule.

The clamp–loader is a hetero-pentameric protein complex. The bacterial clamp–loader is a pentameric complex of one {delta}, one {delta}' and three {gamma} subunits and the bacterial clamp molecule is a homo-dimer of ß-subunits. Bacteriophages have their own complex, the clamp–loader is composed of one gp62 subunit and four gp44 subunits and the clamp is a homo-trimer of gp45 subunits (Trakselis and Benkovic, 2001Go). The eukaryal complex consists of one each of the replication factor C (RFC)1–5 (also called RFCA–E or p140, p40, p36, p37 and p38) subunits (Uhlmann et al., 1996Go). The archaeal complex has one large (RFCL) and four small (RFCS) subunits (Edgell and Doolittle, 1997Go). The eukaryal and archaeal clamp molecules are homo-trimers of PCNA subunits. All of the clamp–loader subunits share AAA+ (or N-terminal and middle) and collar (or C-terminal) domains and are thought to be homologous to each other (Guenther et al., 1997Go; Neuwald et al., 1999Go).

The known architectures are conserved among all of the clamp–loaders from cellular organisms. The three-dimensional structures of bacterial (Escherichia coli) and eukaryal (yeast) clamp–loaders in complex with the cognate clamp molecule were solved by X-ray crystallography (Jeruzalmi et al., 2001Go; Bowman et al., 2004Go) and that of an Archaea (Pyrococcus furiosus) was investigated by the single particle cryo-EM method (Miyata et al., 2004Go). The structure of the P.furiosus RFC small subunit (PfuRFCS) alone was also determined by X-ray crystallography (Oyama et al., 2001Go). The spatial arrangements of the five subunits and their interactions with the clamp molecule basically agree among the three complexes. The five subunits are arranged in a ring shape, in which the collar domains are related by a pseudo-five-fold rotation axis and the AAA+ domains are arranged in a spiral along the pseudo-rotation axis. The clamp molecule interacts with the spiral of the AAA+ domains (Jeruzalmi et al., 2001Go; Bowman et al., 2004Go).

The shared architecture and homology of the clamp–loaders imply a conserved molecular mechanism. On the other hand, the variations in the subunit compositions suggest functional differentiation among the subunits. The different complex versions for each life domain, based on a common architecture, provide an intriguing case to study the molecular evolution of protein machineries.

In this work, the evolutionary processes of archaeal and eukaryal clamp–loader complexes were investigated by using the evolutionary trace (ET) method. ET methods use protein phylogeny to identify functionally important residues. This is usually done by partitioning a molecular phylogenetic tree and search for the residues specifically conserved for a partitioned sequences, which are called as class-specific residues (Lichtarge et al., 1996Go; Lichtarge and Sowa, 2002Go). The class-specific residues were shown to be important for function or structure of proteins in several cases (Lichtarge et al., 1996Go; Shirai and Go, 1997Go; Landgraf et al., 1999Go; Innis et al., 2000Go; Sowa et al., 2001Go; Frenal et al., 2004Go; Shackelford et al., 2004Go; Zhu et al., 2004Go). In this study, an ET method that directly uses inferred ancestral sequences (Shirai et al., 1997Go) has been modified to identify the residues and patterns of replacement responsible for the quarterly structure evolution of the clamp–loaders and clamps.

The results demonstrated that the amino acid replacements during the differentiation process preferentially accumulated at the subunit interfaces. Also, the simpler subunit composition of the archaeal clamp–loader appeared to be a degenerated version of the eukaryal complex, rather than a preserved ancestral type.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Sequences and phylogeny

The amino acid sequences of 60 clamp–loader subunits and 66 clamp subunits were obtained from the EMBL/Genbank/DDBJ (Release 57; Miyazaki et al., 2003Go) and SwissProt (Release 43; Bairoch et al., 2004Go) databases. The sequences were aligned by using the ClustalW program (Higgins et al., 1994Go) and the alignment was manually refined on the XCED alignment editor (Katoh et al., 2002Go). In this study, the residue numbering systems in yeast RFC1-5 and PCNA were based on RFC1_YEAST, RFC2_YEAST, RFC3_YEAST, RFC4_YEAST, RFC5_YEAST and PCNA_YEAST (SwissProt entry codes), respectively. The numbering systems for P.furiosus RFCL, RFCS and PCNA were based on RFCL_PYRFU, RFCS_PYRFU and PCNA_PYRFU, respectively.

A molecular phylogeny was computed by the maximum likelihood method on the PAML application (Yang, 1997Go) with JTT score matrix (Jones et al., 1992Go).

Evolutionary trace (ET)

Ancestral sequences at each node of the phylogenies were inferred by using the PAML application. PAML generated the most likely amino acid types (ancestral sequences) with their likelihoods at each node of phylogeny in the output. The ancestral sequences and the likelihood values were used to devise a score function to rank the inferred amino acid replacements in terms of their significance for the differentiation process. The function was formulated as:

where L(aik) and L(ajk) are the likelihood of the most probable amino acid at site k on nodes i and j, respectively, Maiaj is the score between amino acids ai and aj in the substitution score matrix, Mmax is the highest score in the matrix and Fk is the fraction of branches on which an amino acid replacement at site k has been detected, in the total number of branches in the phylogeny. Sijk takes a value between 0 and 1 and it becomes higher when the inferred replacement at site k on branch ij is more probable (the first term), brings a larger difference in amino acid properties (the second term) and is relatively unique (the third term).

Homology modeling of the PfuRFC–PCNA complex

A homology model of the P.furiosus clamp–loader–clamp (RFC–PCNA) complex was constructed in order to allocate the amino acid replacements detected by the ET on the 3D structure. The structures of the P.furiosus clamp (PfuPCNA) and the small subunit (PfuRFCS) were previously determined by X-ray crystallography (Matsumiya et al., 2001Go; Oyama et al., 2001Go). PfuRFCL was modeled from the crystal structure of yeast RFC1 by using the SwissModel application (Schwede et al., 2003Go). These subunits were assembled by referring to the yeast complex structure (Bowman et al., 2004Go). PfuPCNA was superposed on to PCNA of the yeast complex. The homology model of PfuRFCL was superposed on to yeast RFC1 and the four PfuRFCSs were superposed on to yeast RFC2-5, respectively. Domain orientations in the superposed subunits were manually corrected by fitting to the corresponding domains in the yeast complex. Atomic clashes in the assembled models were removed by repeated cycles of manual modeling, molecular dynamics simulation (1 ps, 300 K, in vacuo simulation by using an AMBER perm 96 force field on the InsightII–Discover application; Accelrys) and an energy minimization.

In the final model, 79, 99 and 100% (cumulatively) of the main chain dihedral angles were found within the favored, allowed and additionally allowed regions of the Ramachandran plot, respectively (plot not shown). The root mean square deviations from the ideal bond geometries were 0.12–0.13 å for bond distances and 1.9–2.3° for bond angles in the final subunit models.

The amino acid replacements detected by ET were mapped on the crystal structure of the yeast complex and on the homology model of the P.furiosus complex, by referring to the sequence alignment. The residues in the models were categorized into interior, surface, interface or ligand sites. Interface sites had accessibility that was reduced by more than 20% in the complex relative to that in the isolated subunit. Ligand sites were in contact with a nucleotide or magnesium ion. Among the remaining residues, those with accessibilities of <0.5 were categorized as interior sites and the others were assigned to surface sites. An in-house program of the Lee and Richards method (Lee and Richards, 1971Go) was used to calculate the accessible surface area.

The difference in the high-Sijk site's distributions on the protein models between subunit differentiation and non-differentiation (speciation) processes was tested with a paired-sample t-test (Campbell, 1974Go). Two-sided t-value was calculated as:

where hd and hn are numbers of branches for differentiation and non-differentiation processes, respectively, md and Vd are the mean and variance of the fraction of sites which fell into a category (surface, interior, interface or ligand), over differentiation branches, respectively, and mn and Vn are the same values for non-differentiation branches.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Archaeal clamp–loader as a degenerated version of the eukaryal complex

One of the most interesting features of the clamp–loader is that the Bacteria, Archaea and Eukarya domains have employed different subunit compositions, in spite of the strongly conserved complex architecture (O'Donnell et al., 2001Go). To search for the origin of these subunit composition differences, a molecular phylogeny was constructed from the amino acid sequences (Figure 1; non-omitted version of phylogeny is available as Supplementary data at PEDS Online).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1. Molecular phylogenies of RFC and PCNA subunits. Note that the branches for species diversification within each subunit cluster (RFC1–5, S, L, Rad, ePCNA and aPCNA) were omitted. The root of RFC phylogeny was inferred by using bacteriophage subunits as the out group. The branches are labeled by the branch number and bootstrap probability (in parentheses) in 1000 reconstructions. The models A, B, E and V are the schemes of the known complexes. The models P1 and P2 are the anticipated ancestral complexes.

 
Bacterial and bacteriophage subunits were initially contained in this analysis. However, because their subunits were distantly related to those of Archaea and Eukarya, they were discarded in order to reduce the uncertainty in the sequence alignment. As an exception, the bacteriophage gp44 subunits were used for the out-group of the phylogeny (Cann et al., 2001Go).

The constructed phylogeny suggested that the archaeal RFCS was not a direct descendant of the ancestral small subunit. If the archaeal clamp–loaders were preserved ancestral type (model P1 in Figure 1), then branch 6 (RFCS-cluster root) should be directly attached to branch 2 (small-subunit cluster-root). This could happen only if branches 2, 4 and 5 were discarded at the same time. Although these branches display relatively lower bootstrap probabilities, the probability of their simultaneous loss is less likely as compared with the other possible combinations that do not support a direct descent of RFCS.

The phylogeny suggested the following evolutional history of the clamp–loader complex. Since the bacterial and bacteriophage subunits did not intervene in this phylogeny, they (models B and V in Figure 1) should have diverged independently from the archaeal and eukaryal subunits. The archaeal and eukaryal subunits first bifurcated into large and small subunits. Although a pentameric complex of one large and four small subunits was expected at this stage (model P1 in Figure 1), the archaeal complex, which adopted the same subunit composition (model A in Figure 1), was not a direct descendant of this complex.

RFC5 next diverged from the small subunit lineage. The anticipated complex at this stage was composed of three types of subunit (model P2 in Figure 1), which would resemble the bacterial complex (model B), if the position of RFC5 relative to RFC1 was conserved. Then, the small subunit lineage diverged into RFC2, -3 and -4, which established the current eukaryal complex (model E in Figure 1).

The archaeal RFCS subunit diverged from the RFC4 lineage after the eukaryal type complex was established. Since no apparent isoform of the small subunits was found in the P.furiosus genome, the genes encoding RFC2, -3 and -5 must have been abolished. This implies that RFCS had to assume the roles of RFC2–5 in this process. This might be a reason for the preservation of RFC4/RFCS lineage in Archaea, that only RFC4 (p40 of human) has clamp-unloading activity by itself (Cai et al., 1997Go).

The PCNA (clamp) molecules of Eukarya and Archaea are homo-trimers. In the molecular phylogeny of PCNA, archaeal subunits (aPCNA in Figure 1) clustered against eukaryal ones (ePCNA in Figure 1; non-omitted version of phylogeny is available as Supplementary data at PEDS Online). The branch 15 in Figure 1, which separates the two domains, should correspond to the process of adaptation of the clamp molecules to each cognate clamp–loader complex.

Interfacial evolution in the subunit differentiation processes

The ancestral sequences at each node in the phylogeny were computed and were used to detect the replacements on each branch. Actually, both the amount and reliability of the replacements varied greatly among the branches. Also, certain neutral replacements complicated the analysis. Therefore, the score system (Sijk) was devised to rank the replacements by their significance. The Sijk score became higher when an anticipated replacement was more likely, brought a larger change in the amino acid properties and was more unique. The influence of an amino acid replacement on protein structure or function is expected to be larger when it occurred at relatively conservative residues and makes a larger change in chemical property of the side chain. Therefore, Sijk is thought to contrast important replacements against nearly neutral ones. Figure 2 shows the profile of an average fraction of sites against the score over all branches in the phylogenies in Figure 1 (including omitted branches). The Sijk values were generally smaller for the branches that were more distantly related to the current proteins, because inferred ancestral sequences were less likely for more ancient proteins. Hence the Sijk values are directly comparable between the sites on the same branch, but between the sites on different branches. Assuming an exponential distribution, the replacements with scores more than one standard deviation higher than the average (over the sites on the same branch) were selected for each branch to normalize the difference in amplitude of Sijk values between branches.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Average of fraction of sites against Sijk score in the RFC and PCNA phylogenies. The plot shows the average of the fraction of sites, which have corresponding Sijk value (horizontal axis), in total number of sites examined (221 and 146 sites for RFC and PCNA subunits, respectively). The average was taken over all branches in the phylogenies. The inset shows the same graph, but with the natural logarithm of fraction for the vertical axis.

 
The high-score sites in the differentiation process of small–large subunits are shown on the yeast complex model (Figure 3a). Branches 1 and 3 in the phylogeny correspond to the large subunit (RFC1) and small subunit (RFC2-5) processes, respectively (Figure 1). Modifications at the interfaces between RFC1 (large) and RFC4 or -5 (small) were expected in these processes. The sites TL4:130T (Sijk = 0.11), LD5:290L (Sijk = 0.23) and IV1:621A (Sijk = 0.10) were found to be responsible for this interface modification. The sites are denoted amino acid in the current protein, before replacement, subunit ID (1–5 for RFC1–5, l and s for RFCL and RFCS and p for PCNA subunits, respectively): residue number in the corresponding numbering system (see Materials and methods) and amino acid after replacement.



View larger version (86K):
[in this window]
[in a new window]
 
Fig. 3. High-Sijk score sites on complex models. (a) The detected sites in the large–small differentiation process on the yeast complex (stereo view). RFC1–5 and PCNA are colored in yellow, sky, magenta, blue, pink and green, respectively. The detected sites are colored in the same, but deeper, colors of the corresponding subunits and are shown in space-filling models. The sites mentioned in the text are labeled A, TL4:130T; B, LD5:290L; C, IV1:621A; and D, VT1:402I. (b) Close-up of the interaction between VT1:402I (RFC1) and L47 and L40 (PCNA). (c) The homologous interaction of that in plate b in the P.furiosus PCNA–PIP peptide complex. (d) The sites in the archaeal subunit differentiation processes on the homology model of P.furiosus RFC–PCNA (stereo view). Subunits are colored the same as the corresponding subunit in the yeast model in plate a. The sites are labeled A, RSs:170K; B, LQs:323L; C, RLl:282R; and D, MAl:306 M. (e) Close-up of the interaction between RSs:170K (RFCS in the position of RFC2) and L262–K264 (RFCS in the position of RFC3). (f) The interaction between LQs:323L (RFCS in the position of RFC2) and V291 (RFCS in the position of RFC5). (g) The detected sites in the PCNA differentiation process. Three PCNA subunits are differently colored to each other. The sites mentioned in the text are labeled A, FPp:125H; B, CRp:81C; C, IAp:147I; and D, LAp:151L. (h) Close-up of the interaction between FPp:125H and K73 (RFC1). (i) Close-up of the interaction among CRp:81C, IAp:147I and LAp:151L (PCNA).

 
A modification at the interface between the clamp–loader and the clamp was also expected in these processes, because the clamp had to be properly oriented to no longer symmetrical clamp–loader. One of the candidates for this modification was VT1:402I (RFC1-V402; Sijk = 0.13), which interacts with L47 and L40 of PCNA (Figure 3b). This is one of the remarkable interactions between RFC1 and PCNA in the yeast complex (Bowman et al., 2004Go). Also, the archaeal PCNA uses a similar hydrophobic interaction with the PIP motif of DNA polymerase {delta} (Figure 3c, Matsumiya et al., 2001Go). This interaction seems to have been established during the large–small differentiation process.

The differentiation of archaeal RFCS is the most intriguing process, because this subunit has been suggested to be a degenerated small subunit. In this process, RFCS had to regain the ability to occupy the positions of RFC2–5, by discarding the specific interactions obtained so far. Considering that combinations of any two of the current five subunits did not show activity (Uhlmann et al., 1996Go), interface rebuilding must have been necessary to make an active complex out of one large and four small subunits.

Branches 10 and 6 correspond to the archaeal RFCL and RFCS processes, respectively (Figure 1). The detected sites are shown on the homology model of the P.furiosus RCF–PCNA complex (Figure 3d). The homology model suggested that RSs:170K (Sijk = 0.35) and LQs:323L (Sijk = 0.23) of RFCS established remarkable interactions in this process, which are still conserved in the current complex (Figure 3e and f). The positively charged side chain of R170 (RS170K) hydrogen bonds with the carbonyl groups at the C-terminal turn of the helix of the neighboring cognate subunit. This interaction should be favored by an electrostatic interaction between the positive charge of R170 (RSs:170K) and the helix dipole (Figure 3e). L323 (LQs:323L) hydrophobically interacts with V291 of the neighboring subunit (Figure 3f). Modifications were also observed in the RFCL subunit (Figure 3d). The sites RLs:282R (Sijk = 0.23) and MAs:306 M (Sijk = 0.45) were found between RFCL and RFCS (placed in the position of RFC4).

This differentiation process between the RFC systems should have been concurrent with the modification of PCNA (clamp) molecules (Figure 3g). Among the replacements on the branch separating archaeal and eukaryal PCNAs (branch 15 in Figure 1), FPp:125H (Sijk = 0.37) was found at the interface between PCNA and RFC. The aromatic side chain of the site is interacting with the alkyl-group of RFC5-K73 in the yeast complex (Figure 3h). CRp:81C (Sijk = 0.78) makes a hydrophobic interaction with IAp:147I (Sijk = 0.64) and LAp:151L (Sijk = 0.72) at the interface between two PCNA subunits (Figure 3i). Note that these replacements are indicated from the Archaea to the Eukarya direction and the interactions are shown on the model of yeast complex. Hence these hydrophobic interactions have been lost in establishing the archaeal complex.

ET as a tool for interface analysis

The above descriptions focused on the remarkable sites at the subunit interfaces. The ability of ET methods to detect protein interfaces has been demonstrated in several cases (Lichtarge and Sowa, 2002Go). Especially when an evolutionary process involved alternation in interaction partners of proteins, the interfaces between domains (Shirai and Go, 1997Go) or between receptor and ligand (Landgraf et al., 1999Go; Innis et al., 2000Go; Sowa et al., 2001Go; Frenal et al., 2004Go; Zhu et al., 2004Go) were detectable by ET methods.

The sites revealed by the method devised in this study appeared to be biased towards the interface or interior regions of the subunits. A total of 173 high-score sites (including those on differentiation processes among large or small subunits, which have not been described in this paper) were detected for the RFC and PCNA subunits; however, most of the sites were not visible on the surface of the complex (Figure 4a and b). This indicates the biased localization of the detected sites. Some of the residue patches at the subunit interfaces were observable when the complex was dissected (Figure 4c).



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 4. Accessible surface view of the yeast RFC–PCNA complex. The models are colored as in Figure 3. (a) A total of 173 high-Sijk sites are highlighted on the surface. (b) Rear view of the same model as plate a. (c) The complex is dissected into RFC1–5 subunits and PCNA trimer to observe the subunit interfaces.

 
The significance of the localization of the high-score sites toward interfaces was tested (Table I). Based on the model structures, all of the residues in the complexes were classified into interior, surface, interface or ligand sites and the distributions of the high-score sites were compared between differentiation and non-differentiation processes. The differentiation processes corresponded to the branches shown in Figure 1 and the non-differentiation (species diversification or speciation) processes corresponded to those omitted in the same phylogenies. As shown in Table I, the distributions were not significantly different for interior or ligand sites between two categories. However, the proportion of interface sites was significantly higher in the differentiation processes, while that of surface sites tended to be lower. This indicates that the high score sites accumulated preferentially at the interface region during subunit differentiation. These results seem to be reasonable, because stoichiometric complex formation is important for the functionality of this protein.


View this table:
[in this window]
[in a new window]
 
Table I. Distribution of high-score sites on molecular regions

 
Analyses of protein interfaces and their contribution to the function of protein machinery are currently an important part of structural biology. Although their applicability to other protein complexes remains to be examined, the results on the clamp–loader complex suggest that the ET method of this study would be useful for the prediction of interface sites on protein subunits.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank Dr K.Morikawa for his advice regarding the manuscript. This work was supported by a research grant endorsed by the New Energy and Industrial Technology Development Organization (NEDO).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bairoch,A., Boeckmann,B., Ferro,S. and Gasteiger,E. (2004) Brief. Bioinform., 5, 39–55.[CrossRef][ISI][Medline]

Bowman,G.D., O'Donnell,M. and Kuriyan,J. (2004) Nature, 429, 724–730.[CrossRef][ISI][Medline]

Cai,J., Gibbs,E., Uhlmann,F., Phillips,B., Yao,N., O'Donnell,M. and Hurwitz,J. (1997) J. Biol. Chem., 272, 18974–18981.[Abstract/Free Full Text]

Campbell,R.C. (1974) Statistics for Biologists, 2nd edn. Cambridge University Press, Cambridge.

Cann,I.K.O., Ishino,S., Yuasa,M., Daiyasu,H., Toh,H. and Ishino,Y. (2001) J. Bacteriol., 183, 2614–2623.[Abstract/Free Full Text]

Edgell,D.R. and Doolittle,W.F. (1997) Cell, 89, 995–998.[CrossRef][ISI][Medline]

Frenal,K., Xu,C.-Q., Wolff,N., Wecker,K., Gurrola,G.B., Zhu,S.-Y., Chi,C.-W., Possani,L.D., Tytgat,J. and Delepierre,M. (2004) Proteins, 56, 367–375.[CrossRef][Medline]

Guenther,B., Onrust,R., Sali,A., O'Donnell,M. and Kuriyan,J. (1997) Cell, 94, 335–345.

Higgins,D., Thompson,J., Gibson,T., Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) Nucleic Acids Res. 22, 4673–4680.[Abstract]

Innis,C.A., Shi,J. and Blundell,T.L. (2000) Protein Eng., 13, 839–847.[CrossRef][ISI][Medline]

Jeruzalmi,D., Yurieva,O., Zhao,Y., Young,M., Stewart,J., Hingorani,M., O'Donnell,M. and Kuriyan,J. (2001) Cell, 106, 417–428.[CrossRef][ISI][Medline]

Jeruzalmi,D., O'Donnell,M. and Kuriyan,J. (2002) Curr. Opin. Struct. Biol., 12, 217–224.[CrossRef][ISI][Medline]

Jones,D.T., Taylor,W.R. and Thornton,J.M. (1992) CABIOS, 8, 275–282.[Medline]

Katoh,K., Misawa,K., Kuma,K. and Miyata, T. (2002) Nucleic Acids Res., 30, 3059–3066.[Abstract/Free Full Text]

Landgraf,R., Fisher,D. and Eisenberg,D. (1999) Protein Eng., 12, 943–951.[CrossRef][ISI][Medline]

Lee,B and Richards,F.M. (1971) J. Mol. Biol., 55, 379–400.[CrossRef][ISI][Medline]

Lichtarge,O. and Sowa,M.E. (2002) Curr. Opin. Struct. Biol., 12, 21–27.[CrossRef][ISI][Medline]

Lichtarge,O., Bourne,H.R. and Cohen,F.E. (1996) J. Mol. Biol., 257, 342–358.[CrossRef][ISI][Medline]

Matsumiya,S., Ishino,Y. and Morikawa,K. (2001) Protein Sci., 10, 17–23.[Abstract/Free Full Text]

Miyata,T., Oyama,T., Mayanagi,K., Ishino,S., Ishino,Y. and Morikawa,K. (2004) Nat. Struct. Mol. Biol., 11, 632–636.[CrossRef][ISI][Medline]

Miyazaki,S., Sugawara,H., Gojobori,T. and Tateno,Y. (2003) Nucleic Acids Res., 30, 13–16.[ISI]

Neuwald,A.F., Aravind,L., Spouge,J.L. and Koonin,E.V. (1999) Genome Res., 9, 27–43.[Abstract/Free Full Text]

O'Donnell, Jeruzalmi,D. and Kuriyan,J. (2001) Curr. Biol., 11, R935–R946.[CrossRef][ISI][Medline]

Oyama T., Ishino,Y., Cann,I.K.O., Ishino,S. and Morikawa,K. (2001) Mol. Cell, 8, 455–463.[CrossRef][ISI][Medline]

Schwede T., Kopp J., Guex N. and Peitsch M.C. (2003) Nucleic Acids Res., 31, 3381–3385.[Abstract/Free Full Text]

Shackelford,G.S., Regni,C.A. and Beamer,L.J. (2004) Protein Sci., 13, 2130–2138.[Abstract/Free Full Text]

Shirai,T. and Go,M. (1997) J. Mol. Evol., 44, S155–S162.[ISI][Medline]

Shirai,T., Suzuki,A., Yamane,T., Ashida,T., Kaobayashi,T., Hitomi,J. and Ito,S. (1997) Protein Eng., 10, 627–634.[CrossRef][ISI][Medline]

Sowa,M.E., He,W., Slep,K.C., Kercher,M.A., Lichtarge,O. and Wensel,T.G. (2001) Nat. Struct. Biol., 8, 234–237.[CrossRef][ISI][Medline]

Stillman,B. (1994) Cell, 78, 725–728.[CrossRef][ISI][Medline]

Trakselis,M.A. and Benkovic,S. (2001) Structure, 9, 999–1004.[CrossRef][ISI][Medline]

Tsurimoto,T. and Stillman,B. (1990) Proc. Natl Acad. Sci. USA, 87, 1023–1027.[Abstract/Free Full Text]

Uhlmann,F., Cai,J., Flores-Rozas,H., Dean,F.B., Finkelstein,J., O'Donnel,M. and Hurwitz,J. (1996) Proc. Natl Acad. Sci. USA, 93, 6521–6526.[Abstract/Free Full Text]

Yang,Z. (1997) CABIOS, 15, 555–556.

Zhu,S., Huys,I., Dyason,K., Verdonck,F. and Tytgat,J. (2004) Proteins, 54, 361–370.[CrossRef][ISI][Medline]

Received November 29, 2004; revised March 5, 2005; accepted March 5, 2005.

Edited by Haruki Nakamura





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
18/3/139    most recent
gzi016v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Saito, M.
Articles by Shirai, T.
PubMed
PubMed Citation
Articles by Saito, M.
Articles by Shirai, T.