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2Ecb Lichtarge Lab 2006 Pages 1–5 2ecb Evolutionary trace report by report maker September 17, 2010 4.3.3 DSSP 4 4.3.4 HSSP 4 4.3.5 LaTex 4 4.3.6 Muscle 4 4.3.7 Pymol 4 4.4 Note about ET Viewer 4 4.5 Citing this work 4 4.6 About report maker 5 4.7 Attachments 5 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2ecb): Title: The solution structure of the third homeobox domain of human zinc fingers and homeoboxes protein Compound: Mol id: 1; molecule: zinc fingers and homeoboxes protein 1; chain: a; fragment: homeobox domain; engineered: yes Organism, scientific name: Homo Sapiens; 2ecb contains a single unique chain 2ecbA (89 residues long). This is an NMR-determined structure – in this report the first model in the file was used. CONTENTS 2 CHAIN 2ECBA 2.1 Q9UKY1 overview 1 Introduction 1 From SwissProt, id Q9UKY1, 80% identical to 2ecbA: 2 Chain 2ecbA 1 Description: Zinc fingers and homeoboxes protein 1. 2.1 Q9UKY1 overview 1 Organism, scientific name: Homo sapiens (Human). 2.2 Multiple sequence alignment for 2ecbA 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.3 Residue ranking in 2ecbA 1 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.4 Top ranking residues in 2ecbA and their position on Catarrhini; Hominidae; Homo. the structure 2 Function: Acts as a transcriptional repressor. 2.4.1 Clustering of residues at 25% coverage. 2 Subunit: Forms homodimers. Also forms heterodimers with ZHX3 2.4.2 Possible novel functional surfaces at 25% which is a prerequisite for repressor activity and with ZHX2. Inter- coverage. 2 acts with NFYA. Interaction: 3 Notes on using trace results 3 Subcellular location: Nuclear. 3.1 Coverage 3 Tissue specificity: Ubiquitously expressed. 3.2 Known substitutions 3 Similarity: Belongs to the ZHX family. 3.3 Surface 3 Similarity: Contains 2 C2H2-type zinc fingers. 3.4 Number of contacts 3 Similarity: Contains 5 homeobox DNA-binding domains. 3.5 Annotation 3 About: This Swiss-Prot entry is copyright. It is produced through a 3.6 Mutation suggestions 3 collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are 4 Appendix 3 no restrictions on its use as long as its content is in no way modified 4.1 File formats 3 and this statement is not removed. 4.2 Color schemes used 4 4.3 Credits 4 2.2 Multiple sequence alignment for 2ecbA 4.3.1 Alistat 4 For the chain 2ecbA, the alignment 2ecbA.msf (attached) with 31 4.3.2 CE 4 sequences was used. The alignment was downloaded from the HSSP 1 Lichtarge lab 2006 Fig. 1. Residues 1-89 in 2ecbA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.) database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 2ecbA.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 31 Total number of residues: 2577 Smallest: 70 Largest: 89 Average length: 83.1 Alignment length: 89 Average identity: 43% Fig. 2. Residues in 2ecbA, colored by their relative importance. Clockwise: Most related pair: 99% front, back, top and bottom views. Most unrelated pair: 14% Most distant seq: 30% Furthermore, 3% of residues show as conserved in this alignment. The alignment consists of 61% eukaryotic ( 54% vertebrata, 3% arthropoda) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 2ecbA.descr. 2.3 Residue ranking in 2ecbA The 2ecbA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2ecbA can be found in the file called 2ecbA.ranks sorted in the attachment. 2.4 Top ranking residues in 2ecbA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 2ecbA colored by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 3. Residues in 2ecbA, colored according to the cluster they belong to: 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the red, followed by blue and yellow are the largest clusters (see Appendix for top 25% of all residues, this time colored according to clusters they the coloring scheme). Clockwise: front, back, top and bottom views. The belong to. The clusters in Fig.3 are composed of the residues listed corresponding Pymol script is attached. in Table 1. Table 1. Table 1. continued cluster size member cluster size member color residues color residues red 17 22,26,29,30,40,44,48,49,51 52,53,54,55,56,58,59,61 continued in next column blue 3 14,16,17 yellow 2 8,9 continued in next column 2 Table 1. continued Table 3. cluster size member res type disruptive color residues mutations 30 F (KE)(TQD)(SNCRG)(M) Table 1. Clusters of top ranking residues in 2ecbA. 58 W (KE)(TQD)(SNCRG)(M) 59 F (KE)(TQD)(SNCRG)(M) 52 R (T)(YVCAG)(SFLWPDI)(EM) 2.4.2 Possible novel functional surfaces at 25% coverage. One 54 E (H)(FW)(Y)(R) group of residues is conserved on the 2ecbA surface, away from (or 61 E (FWH)(Y)(R)(VCAG) susbtantially larger than) other functional sites and interfaces reco- 40 E (FWH)(R)(Y)(VA) gnizable in PDB entry 2ecb. It is shown in Fig. 4. The right panel 49 K (Y)(FW)(T)(SVA) shows (in blue) the rest of the larger cluster this surface belongs to. 29 S (Y)(FW)(KR)(H) 53 R (TYD)(E)(SFVCAWG)(LPI) 51 T (R)(K)(H)(FQW) 56 D (R)(Y)(FW)(H) Table 3. Disruptive mutations for the surface patch in 2ecbA. 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to Fig. 4. A possible active surface on the chain 2ecbA. The larger cluster it some small top percentage of residues [100% is all of the residues belongs to is shown in blue. in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed The residues belonging to this surface ”patch” are listed in Table evolutionary pressure. (I.e., the smaller the coverage, the stronger the 2, while Table 3 suggests possible disruptive replacements for these pressure on the residue.) Starting from the top of that list, mutating a residues (see Section 3.6). couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. Table 2. res type substitutions(%) cvg 3.2 Known substitutions 30 F F(100) 0.03 58 W W(100) 0.03 One of the table columns is “substitutions” - other amino acid types 59 F F(100) 0.03 seen at the same position in the alignment. These amino acid types 52 R R(87)D(12) 0.06 may be interchangeable at that position in the protein, so if one wants 54 E E(83)Q(12)V(3) 0.09 to affect the protein by a point mutation, they should be avoided. For 61 E E(77)N(16)D(6) 0.11 example if the substitutions are “RVK” and the original protein has 40 E E(83)Q(3)D(9) 0.12 an R at that position, it is advisable to try anything, but RVK. Conver- T(3) sely, when looking for substitutions which will not affect the protein, 49 K K(83)Q(3)R(3) 0.14 one may try replacing, R with K, or (perhaps more surprisingly), with G(9) V. The percentage of times the substitution appears in the alignment 29 S S(77)K(3)R(9) 0.16 is given in the immediately following bracket. No percentage is given F(6)Q(3) in the cases when it is smaller than 1%. This is meant to be a rough 53 R R(87)T(3)P(6) 0.18 guide - due to rounding errors these percentages often do not add up K(3) to 100%. 51 T T(80)S(12)P(6) 0.19 3.3 Surface 56 D D(70)H(6)K(12) 0.24 I(6)Q(3) To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 at least 10A˚ , which is roughly the area needed for one water mole- Table 2. Residues forming surface ”patch” in 2ecbA. cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they 3 should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 3.4 Number of contacts COVERAGE Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across V the interface, as well as how many of them are realized through the 100% 50% 30% 5% backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact).
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