Pages 1–6 1iq9 Evolutionary trace report by report maker April 18, 2010 4.3.1 Alistat 5 4.3.2 CE 5 4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 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 1iq9): Title: Crystal structure at 1.8 a of toxin a from naja nigricollis venom Compound: Mol id: 1; molecule: alpha-neurotoxin; chain: a; synonym: short neurotoxin 1 Organism, scientific name: Naja Nigricollis; 1iq9 contains a single unique chain 1iq9A (61 residues long). 2 CHAIN 1IQ9A CONTENTS 2.1 P01426 overview 1 Introduction 1 From SwissProt, id P01426, 100% identical to 1iq9A: Description: Short neurotoxin 1 (Neurotoxin alpha). 2 Chain 1iq9A 1 Organism, scientific name: Naja pallida (Red spitting cobra). 2.1 P01426 overview 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.2 Multiple sequence alignment for 1iq9A 1 Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; 2.3 Residue ranking in 1iq9A 1 Colubroidea; Elapidae; Elapinae; Naja. 2.4 Top ranking residues in 1iq9A and their position on Function: Produces peripheral paralysis by blocking neuromuscular the structure 2 transmission at the postsynaptic site. Binds to the muscular nicotinic 2.4.1 Clustering of residues at 25% coverage. 2 acetylcholine receptor. 2.4.2 Overlap with known functional surfaces at Subcellular location: Secreted. 25% coverage. 2 Tissue specificity: Expressed by the venom gland. 2.4.3 Possible novel functional surfaces at 25% Toxic dose: LD(50) is 0.036 mg/kg by subcutaneous injection. coverage. 3 Similarity: Belongs to the snake toxin family. Type I alpha- neuro- toxin subfamily. 3 Notes on using trace results 4 Caution: The venom of this snake was originally thought to be that 3.1 Coverage 4 of N.nigricollis. 3.2 Known substitutions 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.3 Surface 4 collaboration between the Swiss Institute of Bioinformatics and the 3.4 Number of contacts 4 EMBL outstation - the European Bioinformatics Institute. There are 3.5 Annotation 4 no restrictions on its use as long as its content is in no way modified 3.6 Mutation suggestions 4 and this statement is not removed. 4 Appendix 4 2.2 Multiple sequence alignment for 1iq9A 4.1 File formats 4 For the chain 1iq9A, the alignment 1iq9A.msf (attached) with 291 4.2 Color schemes used 4 sequences was used. The alignment was downloaded from the HSSP 4.3 Credits 5 database, and fragments shorter than 75% of the query as well as 1 Lichtarge lab 2006 Fig. 1. Residues 1-61 in 1iq9A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1iq9A.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 291 Total number of residues: 16901 Smallest: 47 Largest: 61 Average length: 58.1 Alignment length: 61 Average identity: 42% Most related pair: 98% Fig. 2. Residues in 1iq9A, colored by their relative importance. Clockwise: Most unrelated pair: 16% front, back, top and bottom views. Most distant seq: 45% Furthermore, 4% of residues show as conserved in this alignment. The alignment consists of 72% eukaryotic ( 72% 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 1iq9A.descr. 2.3 Residue ranking in 1iq9A The 1iq9A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1iq9A can be found in the file called 1iq9A.ranks sorted in the attachment. 2.4 Top ranking residues in 1iq9A and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 1iq9A 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 1iq9A, 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 15 3,17,23,24,38,39,40,42,43,53 54,56,57,59,60 2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Table 1. Clusters of top ranking residues in 1iq9A. Sulfate ion binding site. Table 2 lists the top 25% of residues at the interface with 1iq9SO4201 (sulfate ion). The following table (Table 2 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 40 C C(99)A 0.08 18/11 3.02 S-S 39 G G(98)AE 0.12 12/12 3.57 V 38 R R(75) 0.20 3/3 4.36 Q(2) L(15)I M(1) F(1)G K(1)WC 24 Y Y(75) 0.21 12/0 2.77 F(19) V(2)CLQ WI Table 2. The top 25% of residues in 1iq9A at the interface with sulfate ion.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each Fig. 4. Residues in 1iq9A, at the interface with sulfate ion, colored by their type in the bracket; noc/bb: number of contacts with the ligand, with the num- relative importance. The ligand (sulfate ion) is colored green. Atoms further ber of contacts realized through backbone atoms given in the bracket; dist: than 30A˚ away from the geometric center of the ligand, as well as on the line distance of closest apporach to the ligand. ) of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1iq9A.) Table 3. res type disruptive mutations 40 C (KER)(QHD)(FYMW)(N) 39 G (R)(K)(EH)(FYQW) 38 R (T)(D)(Y)(E) 24 Y (K)(EQR)(M)(ND) Table 3. List of disruptive mutations for the top 25% of residues in 1iq9A, that are at the interface with sulfate ion. Figure 4 shows residues in 1iq9A colored by their importance, at the interface with 1iq9SO4201. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1iq9A surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1iq9. It is shown in Fig. 5. The residues belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type substitutions(%) cvg antn Fig. 5. A possible active surface on the chain 1iq9A. 53 C C(100) 0.05 S-S 54 C C(100) 0.05 S-S Table 4. continued 59 C C(100) 0.05 S-S 40 C C(99)A 0.08 S-S res type substitutions(%) cvg antn 39 G G(98)AEV 0.12 60 N N(97)D(2)S 0.10 42 C TC(97)SA(1)L 0.13 S-S continued in next column 43 P S(1)P(95)T(1) 0.15 continued in next column 3 Table 4. continued one may try replacing, R with K, or (perhaps more surprisingly), with res type substitutions(%) cvg antn V. The percentage of times the substitution appears in the alignment F(1)N is given in the immediately following bracket. No percentage is given 17 C KC(84)P(6)A(6) 0.18 S-S in the cases when it is smaller than 1%. This is meant to be a rough .(1)RL guide - due to rounding errors these percentages often do not add up 38 R R(75)Q(2)L(15)I 0.20 to 100%. M(1)F(1)GK(1)WC 24 Y Y(75)F(19)V(2)C 0.21 LQWI 3.3 Surface 56 T T(77)S(10)G(1) 0.23 To detect candidates for novel functional interfaces, first we look for K(2)P(1)R(4) residues that are solvent accessible (according to DSSP program) by 2 I(1)NA at least 10A˚ , which is roughly the area needed for one water mole- 57 D D(85)N(8)E(5)QP 0.25 cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor Table 4. Residues forming surface ”patch” in 1iq9A. 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 Table 5. equally important in maintaining the interaction specificity - they res type disruptive should not be automatically dropped from consideration when choo- mutations sing the set for mutagenesis.