Department of Biochemistry and Bioinformatics Centre, Bose Institute,P-1/12, CIT Scheme VIIM, Calcutta 700 054, India
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Abstract |
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Keywords: accessible surface area/molecular recognition/protein-protein complexes/substrate binding/tryptophan
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Introduction |
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In this context, it would be of interest to see if there is any structural or binding feature in the three-dimensional structure of a complex that one can use to distinguish a Trp residue in the hotspot from another which is energetically less important. Such characteristics can then be used to assess the importance of Trp in a protein in the binding of other non-proteinaceous molecules, such as carbohydrate, cofactor, substrate or drug.
We have recently analyzed the environment of Trp residues (the aromatic part of the side chain, in particular) in protein structures, the nature of the interacting residues (partners) and the exponential dependence of the accessible surface area of the Trp residue on its number of partners (other protein residues in contact with Trp) (Samanta et al., 2000). As atoms buried at proteinprotein interfaces are close-packed like the protein interior (Lo Conte et al., 1999
), the aforementioned features of Trp residues in proteins should also be transferable to the residues in the interface region. Consequently, one should be able to assess the role of Trp in the binding by finding the change in the number of its partner residues on complex formation and the associated loss in its accessible surface area and by looking at other elements of its environment and comparing the results with those found within protein structures. This paper is an anatomy of Trp residues in energetically hotspots and other less important regions in proteinprotein interfaces, as well as those involved in the binding of other small molecules.
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Materials and methods |
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Results and discussion |
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Table I lists Trp residues which are/are not in hotspots, as elucidated by Bogan and Thorn (1998). The number of partner residues in contact with the Trp residue, considering either the whole residue or just the aromatic ring, before and after complex formation and the names of the partner residues are also provided. Figure 1
depicts a Trp in the interface and how its partners are disposed in the two subunits.
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Based on the above observations on Trp residues in the protein interface, we wanted to see if it is possible to assess the importance of Trp residues in the binding site of non-proteinaceous molecules in protein structures. For a residue to be important the following two conditions have to be satisfied: ASAw (obs.)
2
ASAw (calc.) and
ASAr (obs.)
2
ASAr (calc.). These conditions are only approximate, as when applied to residues in Table II
, these would have missed out one hotspot residue and also would have identified one non-hotspot residue as important. However, in the case of substrate binding these conditions should be more appropriate. As the substrates are usually much larger than the average size of an amino acid residue and
ASA values are calculated assuming an increase in the number of partners by just one owing to the substrate binding, these values are expected to be smaller than the values actually observed if the Trp residue is crucial for the binding of the substrate. The other criterion for an important residue is the existence of a hydrogen bond between Trp and the substrate molecule.
The formulae of all the substrate molecules used in our analysis and their atoms which are found in contact with the indole ring of Trp residues in different PDB files are shown in Figure 3. Information on Trp residues, their partners, accessible surface areas and how these change on substrate binding is provided in Table III
. In one respect these Trp residues are different from those in the protein interface. Whereas the latter residues have 25 partners (around the aromatic ring) in the parent molecule (Table II
), the majority of the former residues have a value of
6. The substrate molecules are of different shapes and sizes. Trp residues which are deemed to be important in substrate binding using the conditions on ASA are marked with dots in the last column in Table III
. If in addition there is a hydrogen bond between the Trp residue and the substrate, the residue is likely to be important in substrate binding. One example is the binding of FMN by Trp57 in the structure, 1rcf. 1stp corresponds to the structure of streptavidin which binds biotin with exceptionally high affinity (Kd = 1015 M) (Green, 1975
). There are three Trp residues in the binding site (Weber et al., 1989
) and all are shown to be important, thus lending credence to the predictive power of our methodology. Moreover, aromatic-sugar stacking is a typical feature of proteincarbohydrate interactions (Vyas, 1991
; Kadziola et al., 1998
). In all the structures (1byb, 1cel, 1slt and 2gbp) where a carbohydrate molecule is bound, there is at least one Trp residue which is shown to be important. However, in all the cases the decrease in the accessible surface area on substrate binding may not be the best criterion to judge the role of a residue. For example, in the binding of the small sulfate ion (structure, 1sbp), there is hardly any change in ASA and the formation of the hydrogen bond could be the deciding factor in this case. Another situation where the comparison of the observed and calculated values of
ASA may not yield the right result is when the number of partners is atypically small, e.g. 0. In 4fxn, although the observed value is one of the highest in the table, the calculated value is also large and their difference is very small. Nevertheless, this procedure provides some guidelines as to the importance of a Trp residue in binding, which can then be corroborated by protein engineering experiments.
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Depending on the magnitude of contribution towards the binding energy, interface residues have been classified as being or not being in a hotspot (Bogan and Thorn, 1998). In this paper we analyzed whether it is possible to identify Trp residues in hotspots from those which are not, on the basis of crystal structure data. We find that for Trp residues not in hotspots, the change in accessible surface area of the Trp residue on complex formation is restricted to only the indole ring, whereas for hotspot residues the change involves the whole residue. Although the former residues do not form hydrogen bonds with the physiological partner molecule, a hydrogen bond is usually formed for the latter residues. Depending on the change in the number of partner residues, it is possible to calculate the expected change in the accessible surface area of a Trp residue due to complex formation. The observed values are always found to be greater than the calculated values. Similar comparisons between the observed and calculated values and the identification of any hydrogen bond linking Trp to the substrate molecule provides a way to assess the importance of Trp residues in the substrate-binding sites. Based on these encouraging results involving Trp, we are now in the process of extending the methodology to other residues.
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Notes |
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Acknowledgments |
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References |
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Chothia,C. and Janin,J. (1975) Nature, 256, 705708.[ISI][Medline]
Green,N.M. (1975) Adv. Protein Chem., 29, 85133.[Medline]
Hubbard,S.J. (1991) ACCESS, a Program for Calculating Accessibilities. Department of Biochemistry and Molecular Biology, University College London, London.
Janin,J. (1995a) Biochimie, 77, 497505.[ISI][Medline]
Janin,J. (1995b) Proteins: Struct. Funct. Genet., 21, 3039.[ISI][Medline]
Janin,J. and Chothia,C. (1990) J. Biol. Chem., 265, 1602716030.
Jones,S. and Thornton,J.M. (1996) Proc. Natl Acad. Sci. USA, 93, 1320.
Kadziola,A., Sogaard,M., Svensson,B. and Haser,R. (1998) J. Mol. Biol., 278, 205217.[ISI][Medline]
Lee,B. and Richards,F.M. (1971) J. Mol. Biol., 55, 379400.[ISI][Medline]
Lo Conte,L., Chothia,C. and Janin,J. (1999) J. Mol. Biol., 285, 21772198.[ISI][Medline]
McCoy,A.J., Epa,V.A. and Colman,P.M. (1997) J. Mol. Biol., 268, 570584.[ISI][Medline]
Norel,R., Lin,S.L., Wolfson,H.J. and Nussinov,R. (1994) Biopolymers, 34, 933940.[ISI][Medline]
Samanta,U., Pal,D. and Chakrabarti,P. (2000) Proteins: Struct. Funct. Genet., 38, 288300.[ISI][Medline]
Sayle,R.A. and Milner-White,E.J. (1995) Trends Biochem. Sci., 20, 374.[ISI][Medline]
Sussman,J.L., Lin,D., Jiang,J., Manning,N.O., Prilusky,J., Ritter,O. and Abola,E.E. (1998) Acta Crystallogr., D54, 10781084.
Vyas,N.K. (1991) Curr. Opin. Struct. Biol., 1, 732740.
Weber,P.C., Ohlendorf, D.H., Wendoloski,J.J. and Salemme,F.R. (1989) Science, 243, 8588.[ISI][Medline]
Wells,J.A. (1991) Methods Enzymol., 202, 390411.[ISI][Medline]
Received May 9, 2000; revised October 31, 2000; accepted November 9, 2000.