Genome Analysis: Assigning Protein Coding Regions to3D Structures
12

Makiko Suwasalamov@sanger.ac.uk

suwa@hri.co.jp

Asaf A. Salamov

3Christine A. Orengo

orengo@biochem.ucl.ac.jp
12
34

1

Mark B. Swindells

14

swintech@biochem.ucl.ac.jp

Helix Research Institute, 1532-3 Yana, Kisarazu-shi, Chiba, 292, Japan
The

SangerCenter,Wellcome

TrustGenome

Campus,Hinxton,

Cambridge,

CB101SA, UK
Biomolecular Structure and Modelling Unit, Department of Biochemistry, University
College London, Gower Street, London, UK
Inpharmatica Ltd., 60 Charlotte Street, London, W1P2AX, UK1 Introduction

The advent of complete genome sequencing has emphasised the need for comparable developments in
automated analysis, for nding approximate 3D structure of a protein combined with the functional
data. The rst attempt to provide an automated procedure for genome analysis was GeneQuiz [3]andPedant [4], which reported that several ten % of the ORFs have at least one region linked to a known
structure, by using only standard sequence search. Recent approach using fold recognition [5] or PSIblast [8] assigned more high % of ORFs to a known structure. We are interested in maximising thenumber of relationships that can be identi ed on the basis of sequence analysis alone because thesealways precede the use of slower fold-recognition. Furthermore we want to provide a method that canbe immediately applied to all nished genomes. Our work takes advantage of several developments: (1)
CATH database [6]: which classi es homologues on the basis of structural and functional similarities.
(2) The bene ts of using intermediate sequences [2, 7] in sequence searching. (3) PSI-blast: theautomated iterative search techniques [1] which allows more sensitive searches. This work describes anautomated system that uses these developments to maximise the number of sequences that are linked
to a known structure. We rst describe how safe cuto s were determined for the selected sequencealignment methods. Then we show how these methods were applied to 11 microbial genomes.

2 Making a exible system for genome analysis

We determined optimal thresholds for the fasta, gapped-blast and PSI blast. Using several matrices,
fasta and gapped-blast were assessed by linking 841 proteins in in CATH with intermediates fromthe OWL sequence database. First, unrelated pairs were used to determine thresholds, and thenrelated pairs employed to assess the sensitivity. We found that the most sensitive combinations fordatabase searching were fasta/blosum62 and gapped-blast/blosum50, and, normalized thresholds forsafe database searching were found to be 88 and 18, respectively. For PSI-blast, running each test
sequence against a database consisting of both OWL and the 841 test sequences, we found that Evalue thresholds of 0:01 and 0:001 both gave a speci city of around 99%. With these results in hand,
we set about generating a procedure that could analyse large quantities of sequence data.combine asfollowing steps: 1) All sequences from PDB were clustered into families and a representative selected.
2) Using PSI-blast, each PDB representative was run against OWL database. All aligned regions fromsequences below the safer E, were stored together with information about PDB sequence associatedwith each aligned region (psi PDB). 3) For each genome we took the sequences and using the gapped
blast, searched the resulting sequence against psi PDB. Because of the way that psi PDB were made,whatever sequence is hit, a link can be immediately made to a region of a known structure.
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3 Application to 11 genomes

Applying our system, we show how many ORFs could be linked to a PDB le by searching psi PDB
databases. All detailed data are available as an internet solution: Genius (http://www.hri.co.jp/genius).
In this manner, information is available for each hit, when both the PDB/intermediate and intermediate/gene scores are beyond the required thresholds and emphasis that aligned region consisted ofeither >100 residues or >50% of the smaller sequence. In most cases, the results are more than double
those of GeneQuiz and Pedant. For example, we can assign nearly 32% of the M. genitalium ORFs
to a region of known structure. This compares favorably with the results of 12% from GeneQuiz, 16%from Pedant and 22% from frsvr [5]. Although Rychlewski et al. [8], report a higher sensitivity (38%),
one should note that they ran PSI-Blast with a threshold (E < 0:1) without additional checks. It isvaluable to look at the structures which are hit most frequently. We found that vast majority of hits
is ab class structure, which are mostly corresponding to coenzyme. In structural terms, the results
are unusual, because this class is not so major in PDB database. Another major hits correspond toP-loop motifs of ABC transporter, which do not pass the requirement of covering either >50% of
the smaller sequence or >100 residues. It is almost certainly true that many of the P-loops in knownstructures may have arisen from convergent evolution, as their overall topologies can be quite di erent.
Therefore in our opinion, there is currently no clear evidence for a homologous relationship between
ABC transporters and a protein of known structure.

4 Future enhancements

Our automatically generated results are extremely competitive, even without requiring fold recognition
techniques. Genomes can be calculated quickly and updated at regular intervals. In our futureversions we hope to increase the sensitivity by taking full advantage of all the sequences available fromsequencing projects.

References[1] Altschul, S.F., Madden, T.L., Scha er, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J.,
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic
Acids Res., 25:3389{3402, 1997.
[2] Bork, P., Gellerich, J., Groth, H., Hooft, R., and Martin, F., Divergent evolution of a beta/alphabarrel subclass: detection of numerous phospate-binding sites by motif search, Protein Science,
4:268{274, 1995.
[3] Casari, G., Andrade, M.A., Bork, P., Boyle, J., Daruvar, A., Ouzounis, C., Schneider, R.,
Tamames, J., Valencia, A., and Sander, C., Challenging times for bioinformatics, Nature, 376:647-648, 1995.
[4] Frishman, D. and Mewes, H.W., PEDANTic genome analysis, Trends in Genetics, 13:415{416,1997.
[5] Fischer, D. and Eisenberg, D., Assigning folds to proteins encoded by the genome of Mycoplasma
genitalium, Proc. Natl. Acad. Sci. USA, 94:11929{11934, 1997.
[6] Orengo, C.A., Michie, A.D., Jones, S., Jones, D.T., Swindells, M.B., and Thornton, J.M., CATH{a hierarchic classi cation of protein domain structures, Structure, 5:1093-1108, 1997.
[7] Park, J., Teichmann, S.A., Hubbard, T., and Chothia, C., Intermediate sequences increase thedetection of homology between sequences, J. Mol. Biol., 273:349-354, 1997.
[8] Rychlewski, L., Zhang, B. and Godzik, A., Fold and functions for Mycoplasma genitalium proteins,
Folding and Design, 3:229-238, 1998.

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