Sandbox2 Eric Martz
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Does ConSurf-DB Use Too Many Sequences?Does ConSurf-DB Use Too Many Sequences?
In ConSurf-DB, I suppose that no single compromise between too many and too few sequences will produce optimal results for all proteins. Nevertheless, I found the following case informative.
I conclude that perhaps we should caution users: ConSurf-DB is designed to include a wide range of sequences in its multiple-sequence alignments (MSA) and analyses. Often, the MSA will a include substantial number of sequences for proteins with different functions than the query protein. Consequently, amino acids that are colored as highly conserved are truly highly conserved across a wide range of sequence-similar proteins. However, amino acids that are highly conserved in proteins with the same function as the query protein may not appear conserved in ConSurf-DB results. In order to identify these residues, examine the sequences gathered by PSI-BLAST in a ConSurf run, and then set the "Maximum Number of Homologues" equal to the number representing proteins of the same function as the query protein. An example of such an analysis is available.
The example follows.
I was surprised to notice that Q226 in the alpha chain of MHC class I receives a conservation grade of 4 in ConSurf-DB. Q226 is involved in recognizing the CD8 protein, and the binding of CD8 to MHC class I is crucial to MHC class I's function in stimulating T lymphocytes to respond to foreign peptide antigens presented by MHC class I. Therefore I expect Q226 to be highly conserved.
Examining the MSA utilized by ConSurf-DB, I found many proteins other than MHC class I proteins. When I eliminated these, the conservation grade of Q226 went from 4 to 9. Details follow.
Functional Loop 220-230 in MHC Alpha ChainFunctional Loop 220-230 in MHC Alpha Chain
In MHC Class I (mouse and human) Q226 is part of the binding site for CD8[1]. Regarding the alpha chain (A) in human MHC 1akj, the authors state:
A flexible loop of the alpha3 domain (residues 223-229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction ....
Hydrogen bonds between CD8 and the alpha chain of HLA-A2 that involve this loop are (from Table 2[1]):
CD8:D T30.OG1 : T225.O HLA-A2:A 2.7 Å CD8:E S34.OG : Q226.NE2 3.0 CD8:D S100.O : Q226.NE2 3.0 CD8:D S100.OG : Q226.O 2.7 CD8:E Y51.OH : D227.OD2 3.0 CD8:D N99.OD1 : L230.N 3.0 CD8:D N99.ND2 : L230.O 3.4 CD8:D S27.OG1 : E232.OE1 2.7
Similar interactions occur in the mouse in 1bqh[2]. Sequence comparison of the alpha chain of MHC in the CD8-binding regions:
| 195-198 | 220-230 | |
| HLA-A2 | SDHE | D GEDQT QDTEL |
| H-2Kb | PEDK | N GEELI QDMEL |
RED amino acids have conservation grade 8 or 9 for an MSA containing only MHC Class I sequences (136 sequences, see below).
ConSurf Results for 220-230ConSurf Results for 220-230
The first 136* sequences found in Swiss-Prot by a single iteration of PSI-BLAST are for MHC class I molecules from diverse species. After that come non-MHC class I, namely multiple sequences each of Hereditary hemochromatosis protein, Zinc-alpha-2-glycoprotein, IgG receptor FcRn large subunit, and MHC class II (which does not bind to CD8).
| 2VAA chain A | |||||
| Server | Number of Sequences |
Cons. Grades 220-230 |
Av. Cons. Gr. 220-230 |
APD | Job Link |
|---|---|---|---|---|---|
| ConSurf-DB | 144 | 8 57776 48775 | 7.1 | 1.72 | consurfdb |
| ConSurf | all=218 | 8 76565 45553 | 5.9 | 1.17 | 1237248584 |
| ConSurf | 150 | 8 66356 66365 | 6.0 | 0.52 | 1237414642 |
| ConSurf | 139 | 8 98466 98577 | 7.7 | 0.36 | 1237421568 |
| ConSurf | 136* | 8 98466 98587 | 7.8 | 0.33 | 1237421568 |
| ConSurf | 100 | 8 99688 99899 | 9.2 | 0.20 | 1237327837 |
| ConSurf | 70 | 7 99?88 98799 | 8.3 | 0.22 | 1237327964 |
? = insufficient data.
APD = Average Pairwise Difference in the Multiple Sequence Alignment.
ReferencesReferences
- ↑ 1.0 1.1 Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2. Nature. 1997 Jun 5;387(6633):630-4. PMID:9177355 doi:http://dx.doi.org/10.1038/42523
- ↑ Kern PS, Teng MK, Smolyar A, Liu JH, Liu J, Hussey RE, Spoerl R, Chang HC, Reinherz EL, Wang JH. Structural basis of CD8 coreceptor function revealed by crystallographic analysis of a murine CD8alphaalpha ectodomain fragment in complex with H-2Kb. Immunity. 1998 Oct;9(4):519-30. PMID:9806638