1Ytp Lichtarge Lab 2006
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Pages 1–5 1ytp Evolutionary trace report by report maker November 7, 2009 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 4 4.7 Attachments 4 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1ytp): Title: Solution structure of the c4a/c41a variant of the nicotiana alata proteinase inhibitor t1 Compound: Mol id: 1; molecule: proteinase inhibitor; chain: a; fragment: residues 1-53; engineered: yes; mutation: yes Organism, scientific name: Nicotiana Alata; CONTENTS 1ytp contains a single unique chain 1ytpA (53 residues long). This 1 Introduction 1 is an NMR-determined structure – in this report the first model in the file was used. 2 Chain 1ytpA 1 2.1 Q40378 overview 1 2.2 Multiple sequence alignment for 1ytpA 1 2.3 Residue ranking in 1ytpA 1 2.4 Top ranking residues in 1ytpA and their position on 2 CHAIN 1YTPA the structure 1 2.1 Q40378 overview 2.4.1 Clustering of residues at 26% coverage. 1 2.4.2 Possible novel functional surfaces at 26% From SwissProt, id Q40378, 96% identical to 1ytpA: coverage. 2 Description: Proteinase inhibitor. Organism, scientific name: Nicotiana alata (Winged tobacco) (Per- 3 Notes on using trace results 3 sian tobacco). 3.1 Coverage 3 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 3.2 Known substitutions 3 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3.3 Surface 3 eudicots; asterids; lamiids; Solanales; Solanaceae; Nicotiana. 3.4 Number of contacts 3 3.5 Annotation 3 3.6 Mutation suggestions 3 2.2 Multiple sequence alignment for 1ytpA 4 Appendix 3 For the chain 1ytpA, the alignment 1ytpA.msf (attached) with 42 4.1 File formats 3 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 3 database, and fragments shorter than 75% of the query as well as 4.3 Credits 4 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 4 to this report, under the name of 1ytpA.msf. Its statistics, from the 4.3.2 CE 4 alistat program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 1-53 in 1ytpA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.) Format: MSF Number of sequences: 42 Total number of residues: 2193 Smallest: 45 Largest: 53 Average length: 52.2 Alignment length: 53 Average identity: 73% Most related pair: 98% Most unrelated pair: 42% Most distant seq: 68% Furthermore, 15% of residues show as conserved in this alignment. Fig. 2. Residues in 1ytpA, colored by their relative importance. Clockwise: The alignment consists of 50% eukaryotic ( 50% plantae) front, back, top and bottom views. 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 1ytpA.descr. 2.3 Residue ranking in 1ytpA The 1ytpA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1ytpA can be found in the file called 1ytpA.ranks sorted in the attachment. 2.4 Top ranking residues in 1ytpA and their position on the structure In the following we consider residues ranking among top 26% of resi- dues in the protein (the closest this analysis allows us to get to 25%). Figure 2 shows residues in 1ytpA colored by their 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. 2.4.1 Clustering of residues at 26% coverage. Fig. 3 shows the top 26% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed in Table 1. Table 1. Fig. 3. Residues in 1ytpA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for cluster size member the coloring scheme). Clockwise: front, back, top and bottom views. The color residues corresponding Pymol script is attached. red 13 7,8,10,12,13,14,25,27,29,37 41,42,45 susbtantially larger than) other functional sites and interfaces reco- Table 1. Clusters of top ranking residues in 1ytpA. gnizable in PDB entry 1ytp. It is shown in Fig. 4. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 2.4.2 Possible novel functional surfaces at 26% coverage. One 2, while Table 3 suggests possible disruptive replacements for these group of residues is conserved on the 1ytpA surface, away from (or residues (see Section 3.6). 2 Table 2. res type substitutions(%) cvg antn 7 C C(100) 0.15 S-S 8 C C(100) 0.15 S-S 25 C C(100) 0.15 S-S 37 C C(100) 0.15 S-S 45 I I(100) 0.15 12 K K(97)E(2) 0.21 29 S S(97)T(2) 0.21 42 D D(97)N(2) 0.21 41 A C(97)A(2) 0.23 Fig. 4. A possible active surface on the chain 1ytpA. The larger cluster it Table 2. Residues forming surface ”patch” in 1ytpA. belongs to is shown in blue. Table 3. res type disruptive mutations 7 C (KER)(FQMWHD)(NYLPI)(SVA) 8 C (KER)(FQMWHD)(NYLPI)(SVA) 25 C (KER)(FQMWHD)(NYLPI)(SVA) 37 C (KER)(FQMWHD)(NYLPI)(SVA) 45 I (YR)(TH)(SKECG)(FQWD) 12 K (Y)(FW)(T)(VCAG) 29 S (KR)(FQMWH)(NELPI)(Y) 42 D (R)(FWH)(Y)(VCAG) 41 A (KER)(Y)(QHD)(N) Table 3. Disruptive mutations for the surface patch in 1ytpA. 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 couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types may be interchangeable at that position in the protein, so if one wants 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 V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given in the cases when it is smaller than 1%. This is meant to be a rough guide - due to rounding errors these percentages often do not add up to 100%. 3 3.3 Surface 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- cule to come in the contact with the residue. Furthermore, we require COVERAGE that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. V Note, however, that, if our picture of protein evolution is correct, 100% 50% 30% 5% the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) V 3.4 Number of contacts RELATIVE IMPORTANCE Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across the interface, as well as how many of them are realized through the Fig. 5. Coloring scheme used to color residues by their relative importance. backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 1. number of different amino acids appearing in in this column 3.5 Annotation of the alignment If the residue annotation is available (either from the pdb file or 2. their type from other sources), another column, with the header “annotation” • rho 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 bond forming residue), and sb (for salt bridge forming residue).