Published online before print
December 1, 2005, 10.1110/ps.051882006
Protein Science (2006), 15:208-210.
Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
FOR THE RECORD
Structural interpretation of mutations and SNPs using STRAP-NT
Christoph Gille
Institute of Biochemistry Charité, Medical Faculty of the Humboldt University, 10117 Berlin, Germany
Reprint requests to: Christoph Gille, Institute of Biochemistry Charité, Medical Faculty of the Humboldt University, 10117 Berlin, Monbijoustrasse 2, Germany; e-mail: christoph.gille{at}charite.de; fax: +49-30-450528942.
(RECEIVED October 5, 2005;
FINAL REVISION October 12, 2005;
ACCEPTED October 13, 2005)
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Abstract
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Visualization of residue positions in protein alignments and mapping onto suitable structural models is an important first step in the interpretation of mutations or polymorphisms in terms of protein function, interaction, and thermodynamic stability. Selecting and highlighting large numbers of residue positions in a protein structure can be time-consuming and tedious with currently available software. Previously, a series of tasks and analyses had to be performed one-by-one to map mutations onto 3D protein structures; STRAP-NT is an extension of STRAP that automates these tasks so that users can quickly and conveniently map mutations onto 3D protein structures. When the structure of the protein of interest is not yet available, a related protein can frequently be found in the structure databases. In this case the alignment of both proteins becomes the crucial part of the analysis. Therefore we embedded these program modules into the Java-based multiple sequence alignment program STRAP-NT. STRAP-NT can simultaneously map an arbitrary number of mutations denoted using either the nucleotide or amino acid sequence. When the designations of the mutations refer to genomic sites, STRAP-NT translates them into the corresponding amino acid positions, taking intron–exon boundaries into account. STRAP-NT tightly integrates a number of current protein structure viewers (currently PYMOL, RASMOL, JMOL, and VMD) with which mutations and polymorphisms can be directly displayed on the 3D protein structure model. STRAP-NT is available at the PDB site and at http://www.charite.de/bioinf/strap/ or http://strapjava.de.
Keywords: protein structure/folding; 3D structure; genotype/phenotype relationship; structure visualization; mutation screening; mutations/SNPs
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051882006.
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Introduction
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Studying the effects of amino acid exchanges in proteins often involves mapping onto a three-dimensional (3D) structure (Stitziel et al. 2003). This is usually time-consuming because it requires several manual steps: If structural data for the protein of interest is not available, a close relative to the protein with a known 3D structure must be identified. A sequence search against the Protein Data Bank (PDB) is usually performed to find a suitable candidate. The key step of the entire process is the alignment of both proteins to assign the selected residues of the protein being analyzed to residues of the protein with the known structure. Finally, the residues are highlighted in the structure either by choosing a different representation or by attaching labels. In the protein structure record, each residue is specified by a number. This numbering does not necessarily start from 1 and may skip numbers, and consequently differs to the residue index. To facilitate the visualization of residue positions in protein structures, we redesigned and extended STRAP, an interactive and extendable alignment program for protein sequences (Gille and Frömmel 2001).
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Results and Discussion
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A program package was created that allows mapping of amino acid or nucleotide positions onto 3D protein structures. In the following, we present an example of how to use STRAP-NT to perform such an analysis using a novel missense mutation of the 
cardiac myosin heavy chain (MYHC7, GenBank file M57965
[GenBank]
) owing to a cytosine-to-thymine substitution at position 12164 of the genomic nucleotide sequence (Daehmlow et al. 2002). Since almost all previously described mutations found in the MYHC7 were associated with hypertrophic cardiomyopathy (HCM), the identification of this mutation in a patient suffering from dilated cardiomyopathy (DCM) was surprising. We addressed the question, What structural features distinguish C12164T from mutations associated with HCM? The first step in our analysis was to select all amino acid positions in the protein sequence of MYHC7 where mutations had been described. The mutation data was collected from several Web sources, including databases, articles, and abstracts. STRAP-NT readily imports lists of changes on the nucleotide sequence level as well as on the amino acid sequence level (Antonarakis 1998). STRAP-NT displays each imported mutation in the MYHC7 at the corresponding position in the sequence. The translation into a sequence of amino acids shows that the nucleotide exchange C12164T is a missense mutation leading to the substitution of Ser at position 642 by Leu. Because no structural information was available for cardiac myosin, we used the PDB entry 1alm containing myosin and actin from skeletal muscle. Cardiac myosin and striated muscle myosin were automatically aligned in STRAP-NT (Fukami-Kobayashi and Saito 2002). Subsequently all positions selected in cardiac myosin were automatically mapped onto skeletal muscle myosin. By underlining all mutations in the 3D structure, a spatial feature of the Ser642Leu mutation became evident: The mutation Ser642Leu of the patient with DCM was located at the interface to actin, whereas the mutations associated with HCM were located in other regions of myosin. This feature is shared by another previously described mutation Ser532Pro of a patient with DCM that is located very close to the mutation Ser642Leu in the interface of myosin to actin (Kamisago et al. 2000). These results suggest that mutations in myosin located in the interface to actin cause DCM, whereas other mutations induce HCM. This example shows the usage of the software for mutations and polymorphisms in proteins. Another useful application is the highlighting of certain sites that are given in scientific texts and publications in multiple sequence alignments or in 3D structures. This example demonstrates that mapping of mutations onto 3D structures essentially requires user interaction at several stages and cannot be performed completely automatically. The pivotal step is the choice of a suitable 3D template and its alignment to the protein that bears the mutations. Sequence alignments require inspection and manual refinement (Stitziel et al. 2004), particularly when both sequences differ markedly. Internet-based services such as ClustalW (Fukami-Kobayashi and Saito 2002) are available with which individual tasks in the above describe analysis can be performed, but a significant amount of user intervention is required. Internet servers for individual proteins are available, but users are restricted to the proteins and mutations offered by those services (Stitziel et al. 2004). In contrast, STRAP-NT allows users to analyze arbitrary mutations and proteins. STRAP-NT has several interactive tutorials that take new users step-by-step through a series of analyses, including the one described here. Each tutorial can be completed within ~90 min. To our knowledge STRAP-NT is currently the only available program for mapping mutations that are given as indices of nucleotides or amino acids onto 3D models. In the light of the major changes we added the suffix "-NT" to the package name to emphasize the importance of genomic organization, reading frames, and splice iso-forms.
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Figure 1. Flow chart of the analysis of mutations. The analysis requires user interaction at several steps that are enclosed in simple boxes in the chart. Those steps that run automatically are contained in double-frame boxes. External programs embedded in STRAP like ClustalW and Pymol are indicated at the button of the boxes. This flow chart was prepared with the LaTeX style Flow.sty by Kees Lindhout and Leo van Geest.
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References
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Antonarakis, S. E. 1998. Recommendations for a nomenclature system for human gene mutations. Nomenclature working group. Hum. Mutat. 11: 1–3.[CrossRef][Medline]Daehmlow, S., Erdmann, J. Knueppel, T., Gille, C., Frömmel, C., Hummel, M., Hetzer, R., and Regitz-Zagrosek, V. 2002. Novel mutations in sarcomeric protein genes in dilated ardiomyopathy. Biochem. Biophys. Res. Commun. 298: 116–120.[CrossRef][Medline]
Fukami-Kobayashi, K. and Saito, N. 2002. How to make good use of CLUSTALW. Tanpakushitsu Kakusan Koso 47: 1237–1239.[Medline]
Gille, C. and Frömmel, C. 2001. Strap: Editor for structural alignments of proteins. Bioinformatics 17: 377–378.[Abstract/Free Full Text]
Kamisago, M., Sharma, S.D., DePalma, S.R., Solomon, S., Sharma, P., McDonough, B., Smoot, L., Mullen, M.P., Woolf, P.K., Wigle, E.D., et al. 2000. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N. Engl. J. Med. 343: 1688–1696.[Abstract/Free Full Text]
Stitziel, N.O., Tseng, Y.Y., Pervouchine, D., Goddeau, D., Kasif, S., and Liang, J. 2003. Structural location of disease-associated single-nucleotide polymorphisms. J. Mol. Biol. 327: 1021–1030.[CrossRef][Medline]
Stitziel, N.O., Binkowski, T.A., Tseng, Y.Y., Kasif, S., and Liang, J. 2004. Toposnp: A topographic database of non-synonymous single nucleotide polymorphisms with and without known disease association. Nucleic Acids Res. 32: 520–522.
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Abstract
Introduction
