Pages 1–4 2h3m Evolutionary trace report by report maker September 18, 2008 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 4 4.7 Attachments 4 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2h3m): Title: Crystal structure of zo-1 pdz1 Compound: Mol id: 1; molecule: tight junction protein zo-1; chain: a; fragment: first pdz domain; synonym: zonula occludens 1 protein, zona occludens 1 protein, tight junction protein 1; engineered: yes Organism, scientific name: Homo Sapiens; 2h3m contains a single unique chain 2h3mA (94 residues long). 2 CHAIN 2H3MA 2.1 Q4ZGJ6 overview From SwissProt, id Q4ZGJ6, 100% identical to 2h3mA: CONTENTS Description: Tight junction protein 1 (Zona occludens 1). Organism, scientific name: Homo sapiens (Human). 1 Introduction 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2 Chain 2h3mA 1 Catarrhini; Hominidae; Homo. 2.1 Q4ZGJ6 overview 1 2.2 Multiple sequence alignment for 2h3mA 1 2.2 Multiple sequence alignment for 2h3mA 2.3 Residue ranking in 2h3mA 1 For the chain 2h3mA, the alignment 2h3mA.msf (attached) with 6 2.4 Top ranking residues in 2h3mA and their position on sequences was used. The alignment was assembled through combi- the structure 1 nation of BLAST searching on the UniProt database and alignment 2.4.1 Clustering of residues at 37% coverage. 1 using Muscle program. It can be found in the attachment to this report, under the name of 2h3mA.msf. Its statistics, from the alistat 3 Notes on using trace results 2 program are the following: 3.1 Coverage 2 3.2 Known substitutions 2 Format: MSF 3.3 Surface 2 Number of sequences: 6 3.4 Number of contacts 2 Total number of residues: 554 3.5 Annotation 3 Smallest: 89 3.6 Mutation suggestions 3 Largest: 94 Average length: 92.3 4 Appendix 3 Alignment length: 94 4.1 File formats 3 Average identity: 66% 4.2 Color schemes used 3 Most related pair: 99% 4.3 Credits 3 Most unrelated pair: 52% 4.3.1 Alistat 3 Most distant seq: 66% 4.3.2 CE 3 4.3.3 DSSP 3 4.3.4 HSSP 3 Furthermore, 37% of residues show as conserved in this alignment. 1 Lichtarge lab 2006 Fig. 1. Residues 17-110 in 2h3mA colored by their relative importance. (See Appendix, Fig.4, for the coloring scheme.) The alignment consists of 83% eukaryotic ( 83% vertebrata) sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 2h3mA.descr. 2.3 Residue ranking in 2h3mA The 2h3mA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2h3mA can be found in the file called 2h3mA.ranks sorted in the attachment. 2.4 Top ranking residues in 2h3mA and their position on the structure In the following we consider residues ranking among top 37% Fig. 3. Residues in 2h3mA, colored according to the cluster they belong to: of residues in the protein (the closest this analysis allows us to red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The get to 25%). Figure 2 shows residues in 2h3mA colored by their corresponding Pymol script is attached. importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1. Table 1. cluster size member color residues red 26 25,26,33,34,35,36,37,39,40 41,43,53,57,58,59,64,65,67 69,78,79,90,91,95,100,102 blue 7 19,20,21,23,73,105,108 Table 1. Clusters of top ranking residues in 2h3mA. 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 some small top percentage of residues [100% is all of the residues 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 evolutionary pressure. (I.e., the smaller the coverage, the stronger the pressure on the residue.) Starting from the top of that list, mutating a Fig. 2. Residues in 2h3mA, colored by their relative importance. Clockwise: couple of residues should affect the protein somehow, with the exact front, back, top and bottom views. effects to be determined experimentally. 3.2 Known substitutions 2.4.1 Clustering of residues at 37% coverage. Fig. 3 shows the One of the table columns is “substitutions” - other amino acid types top 37% of all residues, this time colored according to clusters they seen at the same position in the alignment. These amino acid types belong to. The clusters in Fig.3 are composed of the residues listed may be interchangeable at that position in the protein, so if one wants 2 to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with COVERAGE V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given V in the cases when it is smaller than 1%. This is meant to be a rough 100% 50% 30% 5% guide - due to rounding errors these percentages often do not add up to 100%. 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by V 2 at least 10A˚ , which is roughly the area needed for one water mole- RELATIVE IMPORTANCE 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. Fig. 4. Coloring scheme used to color residues by their relative importance. 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 4 APPENDIX should not be automatically dropped from consideration when choo- 4.1 File formats sing the set for mutagenesis. (Especially if they form a cluster with Files with extension “ranks sorted” are the actual trace results. The the surface residues.) fields in the table in this file: 3.4 Number of contacts • alignment# number of the position in the alignment Another column worth noting is denoted “noc/bb”; it tells the num- • residue# residue number in the PDB file ber of contacts heavy atoms of the residue in question make across • type amino acid type the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, • rank rank of the position according to older version of ET mutation presumably won't have strong impact). Two heavy atoms • variability has two subfields: are considered to be “in contact” if their centers are closer than 5A˚ . 1. number of different amino acids appearing in in this column of the alignment 3.5 Annotation 2. their type If the residue annotation is available (either from the pdb file or • rho from other sources), another column, with the header “annotation” ET score - the smaller this value, the lesser variability of appears. Annotations carried over from PDB are the following: site this position across the branches of the tree (and, presumably, (indicating existence of related site record in PDB ), S-S (disulfide the greater the importance for the protein) bond forming residue), hb (hydrogen bond forming residue, jb (james • cvg coverage - percentage of the residues on the structure which bond forming residue), and sb (for salt bridge forming residue). have this rho or smaller • gaps 3.6 Mutation suggestions percentage of gaps in this column Mutation suggestions are completely heuristic and based on comple- 4.2 Color schemes used mentarity with the substitutions found in the alignment. Note that The following color scheme is used in figures with residues colored they are meant to be disruptive to the interaction of the protein by cluster size: black is a single-residue cluster; clusters composed of with its ligand. The attempt is made to complement the following more than one residue colored according to this hierarchy (ordered properties: small [AV GST C], medium [LP NQDEMIK], large by descending size): red, blue, yellow, green, purple, azure, tur- [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, tively [KHR], or negatively [DE] charged, aromatic [W F Y H], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, long aliphatic chain [EKRQM], OH-group possession [SDET Y ], DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, and NH2 group possession [NQRK]. The suggestions are listed tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. according to how different they appear to be from the original amino The colors used to distinguish the residues by the estimated acid, and they are grouped in round brackets if they appear equally evolutionary pressure they experience can be seen in Fig.