1Cxn Lichtarge Lab 2006

Pages 1–5 1cxn Evolutionary trace report by report maker December 10, 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 5 4.5 Citing this work 5 4.6 About report maker 5 4.7 Attachments 5 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1cxn): Title: Refined three-dimensional solution structure of a snake cardiotoxin: analysis of the side-chain organisation suggests the existence of a possible phospholipid binding site Compound: Mol id: 1; molecule: cardiotoxin gamma; chain: a; engineered: yes Organism, scientific name: Naja Nigricollis 1cxn contains a single unique chain 1cxnA (60 residues long). This is an NMR-determined structure – in this report the first model in the file was used. CONTENTS 2 CHAIN 1CXNA 1 Introduction 1 2.1 P01468 overview 2 Chain 1cxnA 1 From SwissProt, id P01468, 100% identical to 1cxnA: 2.1 P01468 overview 1 Description: Cytotoxin 1 (Cardiotoxin gamma). 2.2 Multiple sequence alignment for 1cxnA 1 Organism, scientific name: Naja pallida (Red spitting cobra). 2.3 Residue ranking in 1cxnA 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.4 Top ranking residues in 1cxnA and their position on Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; the structure 2 Colubroidea; Elapidae; Elapinae; Naja. 2.4.1 Clustering of residues at 25% coverage. 2 Function: Shows cytolytic activity (By similarity). 2.4.2 Possible novel functional surfaces at 25% Subcellular location: Secreted. coverage. 2 Tissue specificity: Expressed by the venom gland. Similarity: Belongs to the snake toxin family. Type IA cytotoxin 3 Notes on using trace results 3 subfamily. 3.1 Coverage 3 Caution: The venom of this snake was originally thought to be that 3.2 Known substitutions 3 of N.nigricollis while it is really from N.pallida. 3.3 Surface 3 About: This Swiss-Prot entry is copyright. It is produced through a 3.4 Number of contacts 3 collaboration between the Swiss Institute of Bioinformatics and the 3.5 Annotation 3 EMBL outstation - the European Bioinformatics Institute. There are 3.6 Mutation suggestions 3 no restrictions on its use as long as its content is in no way modified and this statement is not removed. 4 Appendix 4 4.1 File formats 4 2.2 Multiple sequence alignment for 1cxnA 4.2 Color schemes used 4 For the chain 1cxnA, the alignment 1cxnA.msf (attached) with 213 4.3 Credits 4 sequences was used. The alignment was downloaded from the HSSP 4.3.1 Alistat 4 database, and fragments shorter than 75% of the query as well as 4.3.2 CE 4 duplicate sequences were removed. It can be found in the attachment 1 Lichtarge lab 2006 Fig. 1. Residues 1-60 in 1cxnA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.) to this report, under the name of 1cxnA.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 213 Total number of residues: 12094 Smallest: 46 Largest: 60 Average length: 56.8 Alignment length: 60 Average identity: 45% Most related pair: 98% Most unrelated pair: 20% Fig. 2. Residues in 1cxnA, colored by their relative importance. Clockwise: Most distant seq: 42% front, back, top and bottom views. Furthermore, 6% of residues show as conserved in this alignment. The alignment consists of 71% eukaryotic ( 71% 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 1cxnA.descr. 2.3 Residue ranking in 1cxnA The 1cxnA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1cxnA can be found in the file called 1cxnA.ranks sorted in the attachment. 2.4 Top ranking residues in 1cxnA 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 1cxnA 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 25% coverage. Fig. 3 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed Fig. 3. Residues in 1cxnA, colored according to the cluster they belong to: in Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The Table 1. corresponding Pymol script is attached. cluster size member color residues red 14 14,21,36,37,38,39,42,43,53 54,56,57,59,60 2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1cxnA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- Table 1. Clusters of top ranking residues in 1cxnA. gnizable in PDB entry 1cxn. It is shown in Fig. 4. The residues belonging to this surface ”patch” are listed in Table 2, while Table 2 Table 3. continued res type disruptive mutations 59 C (KER)(FQMWHD)(NLPI)(Y) 43 P (R)(Y)(H)(K) 42 C (K)(ER)(QM)(D) 37 G (R)(K)(H)(FW) 14 C (FW)(EHR)(KYD)(M) 60 N (Y)(FWH)(R)(T) 36 R (TYD)(E)(S)(CG) 17 G (R)(K)(E)(H) 56 T (FW)(R)(KH)(M) 39 I (R)(Y)(H)(K) 57 D (R)(FWH)(Y)(VCAG) Table 3. Disruptive mutations for the surface patch in 1cxnA. 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Fig. 4. A possible active surface on the chain 1cxnA. Trace results are commonly expressed in terms of coverage: the residue is important if its “coverage” is small - that is if it belongs to 3 suggests possible disruptive replacements for these residues (see some small top percentage of residues [100% is all of the residues Section 3.6). 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 Table 2. to some nearby percentage), sorted by the strength of the presumed res type substitutions(%) cvg antn evolutionary pressure. (I.e., the smaller the coverage, the stronger the 38 C C(100) 0.07 S-S pressure on the residue.) Starting from the top of that list, mutating a 53 C C(100) 0.07 S-S couple of residues should affect the protein somehow, with the exact 54 C C(100) 0.07 S-S effects to be determined experimentally. 59 C C(99). 0.08 S-S 43 P GP(98)TA 0.10 3.2 Known substitutions 42 C C(99)Y 0.12 S-S One of the table columns is “substitutions” - other amino acid types 37 G G(98)E(1)A 0.13 seen at the same position in the alignment. These amino acid types 14 C QC(92)KT(6) 0.15 S-S may be interchangeable at that position in the protein, so if one wants 60 N N(98)S.D 0.17 to affect the protein by a point mutation, they should be avoided. For 36 R R(84)L(12)M(2)G 0.18 example if the substitutions are “RVK” and the original protein has 17 G WG(85)A(8)V(1). 0.20 an R at that position, it is advisable to try anything, but RVK. Conver- SD(1)E sely, when looking for substitutions which will not affect the protein, 56 T T(86)K(2)G(1)N 0.22 one may try replacing, R with K, or (perhaps more surprisingly), with S(7)R(1) V. The percentage of times the substitution appears in the alignment 39 I T(9)A(24)I(32) 0.23 is given in the immediately following bracket. No percentage is given V(1)G(30)S in the cases when it is smaller than 1%. This is meant to be a rough 57 D D(83)N(12)E(4) 0.25 guide - due to rounding errors these percentages often do not add up to 100%. Table 2. Residues forming surface ”patch” in 1cxnA. 3.3 Surface Table 3. To detect candidates for novel functional interfaces, first we look for res type disruptive residues that are solvent accessible (according to DSSP program) by A˚ 2 mutations at least 10 , which is roughly the area needed for one water mole- 38 C (KER)(FQMWHD)(NYLPI)(SVA) cule to come in the contact with the residue. Furthermore, we require 53 C (KER)(FQMWHD)(NYLPI)(SVA) that these residues form a “cluster” of residues which have neighbor A˚ 54 C (KER)(FQMWHD)(NYLPI)(SVA) within 5 from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, continued in next column 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 number 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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