Pages 1–9 1h9p Evolutionary trace report by report maker August 28, 2010 4.3.1 Alistat 8 4.3.2 CE 8 4.3.3 DSSP 8 4.3.4 HSSP 8 4.3.5 LaTex 8 4.3.6 Muscle 8 4.3.7 Pymol 8 4.4 Note about ET Viewer 8 4.5 Citing this work 8 4.6 About report maker 9 4.7 Attachments 9 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1h9p): Title: Crystal structure of dioclea guianensis seed lectin Compound: Mol id: 1; molecule: seed lectin; chain: a Organism, scientific name: Dioclea Guianensis; 1h9p contains a single unique chain 1h9pA (233 residues long). 2 CHAIN 1H9PA 2.1 P81637 overview CONTENTS From SwissProt, id P81637, 91% identical to 1h9pA: Description: Lectin alpha chain [Contains: Lectin beta chain; Lectin 1 Introduction 1 gamma-1 chain; Lectin gamma-2 chain]. 2 Chain 1h9pA 1 Organism, scientific name: Dioclea guianensis. 2.1 P81637 overview 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.2 Multiple sequence alignment for 1h9pA 1 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 2.3 Residue ranking in 1h9pA 1 eudicotyledons; rosids; eurosids I; Fabales; Fabaceae; Papilionoi- 2.4 Top ranking residues in 1h9pA and their position on deae; Phaseoleae; Dioclea. the structure 1 Function: D-mannose/D-glucose-binding lectin. Mixture of 60lectin 2.4.1 Clustering of residues at 25% coverage. 2 and 40 2.4.2 Overlap with known functional surfaces at Subunit: Equilibrium between homodimer and homotetramer. 25% coverage. 3 Ptm: The beta and gamma chains are produced by partial proteolytic 2.4.3 Possible novel functional surfaces at 25% processing of the lectin alpha chain by an asparaginyl endopeptidase. coverage. 5 Mass spectrometry: MW=25398; MW ERR=1; METHOD=Electrospray; RANGE=1-237; NOTE=Ref.1. 3 Notes on using trace results 7 Mass spectrometry: MW=12831; MW ERR=1; 3.1 Coverage 7 METHOD=Electrospray; RANGE=1-118; NOTE=Ref.1. 3.2 Known substitutions 7 Mass spectrometry: MW=12583; MW ERR=1; 3.3 Surface 7 METHOD=Electrospray; RANGE=119-237; NOTE=Ref.1. 3.4 Number of contacts 7 Mass spectrometry: MW=12012; MW ERR=1; 3.5 Annotation 7 METHOD=Electrospray; RANGE=125-237; NOTE=Ref.1. 3.6 Mutation suggestions 7 Miscellaneous: Binds one manganese (or other transition metal) ion and one calcium ion. The metal ions are essential for the saccharide- 4 Appendix 7 binding and cell-agglutinating activities. 4.1 File formats 7 Similarity: Belongs to the leguminous lectin family. 4.2 Color schemes used 8 About: This Swiss-Prot entry is copyright. It is produced through a 4.3 Credits 8 collaboration between the Swiss Institute of Bioinformatics and the 1 Lichtarge lab 2006 in 1h9pA can be found in the file called 1h9pA.ranks sorted in the attachment. 2.4 Top ranking residues in 1h9pA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1h9pA colored by their importance: bright red and yellow indicate more conser- Fig. 1. Residues 1-116 in 1h9pA colored by their relative importance. (See ved/important residues (see Appendix for the coloring scheme). A Appendix, Fig.11, for the coloring scheme.) Pymol script for producing this figure can be found in the attachment. Fig. 2. Residues 117-237 in 1h9pA colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.) EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 2.2 Multiple sequence alignment for 1h9pA For the chain 1h9pA, the alignment 1h9pA.msf (attached) with 271 sequences was used. The alignment was downloaded from the HSSP 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 1h9pA.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 1h9pA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 271 Total number of residues: 59472 Smallest: 175 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 233 top 25% of all residues, this time colored according to clusters they Average length: 219.5 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 233 in Table 1. Average identity: 40% Most related pair: 99% Table 1. Most unrelated pair: 17% cluster size member Most distant seq: 33% color residues red 57 6,7,8,9,10,11,24,26,28,29,30 34,50,52,54,61,81,89,92,93 Furthermore, <1% of residues show as conserved in this ali- 94,97,98,108,109,110,111,113 gnment. 128,133,154,156,157,171,172 The alignment consists of 66% eukaryotic ( 66% plantae) 173,175,178,181,182,189,190 sequences. (Descriptions of some sequences were not readily availa- 191,193,195,197,199,207,208 ble.) The file containing the sequence descriptions can be found in 209,212,213,214,230,231,232 the attachment, under the name 1h9pA.descr. 233 2.3 Residue ranking in 1h9pA Table 1. Clusters of top ranking residues in 1h9pA. The 1h9pA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues 2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) QFR Table 2. The top 25% of residues in 1h9pA at the interface with cad- mium 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 type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Table 3. res type disruptive mutations 10 D (R)(H)(FW)(Y) 8 E (H)(Y)(FW)(R) 24 H (E)(D)(M)(Q) 34 S (R)(K)(H)(Y) Table 3. List of disruptive mutations for the top 25% of residues in Fig. 4. Residues in 1h9pA, colored according to the cluster they belong to: 1h9pA, that are at the interface with cadmium ion. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. 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. Cadmium ion binding site. Table 2 lists the top 25% of residues at the interface with 1h9pACD238 (cadmium ion). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 10 D D(91) 0.05 5/1 2.05 site N(3) V(1)GF L(1)EIS 8 E E(85) 0.07 4/0 2.29 site V(8) A(4)DNQ F 24 H H(82) 0.10 5/0 2.30 R(7) .(4)LA Fig. 5. Residues in 1h9pA, at the interface with cadmium ion, colored by D(1) their relative importance. The ligand (cadmium ion) is colored green. Atoms Q(1)CSG further than 30A˚ away from the geometric center of the ligand, as well as on 34 S S(81) 0.10 1/0 4.21 the line of sight to the ligand were removed. (See Appendix for the coloring P(9)X scheme for the protein chain 1h9pA.) W(2) I(2)TLD continued in next column Figure 5 shows residues in 1h9pA colored by their importance, at the interface with 1h9pACD238. Cadmium ion binding site. Table 4 lists the top 25% of residues at the interface with 1h9pACD239 (cadmium ion). The following table 3 (Table 5) suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 10 D D(91) 0.05 4/0 2.26 site N(3) V(1)GF L(1)EIS 24 H H(82) 0.10 1/0 4.58 R(7) .(4)LA D(1) Q(1)CSG 208 D D(71) 0.21 2/1 4.61 Y(8) S(2) H(4)Q V(3) E(2)N T(1) A(1) Fig. 6. Residues in 1h9pA, at the interface with cadmium ion, colored by F(1)IG their relative importance. The ligand (cadmium ion) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on Table 4. The top 25% of residues in 1h9pA at the interface with cad- the line of sight to the ligand were removed. (See Appendix for the coloring mium ion.(Field names: res: residue number in the PDB entry; type: amino scheme for the protein chain 1h9pA.) acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; substs: substitutions seen in the alignment; with the percentage of each type dist: distance of closest apporach to the ligand. ) in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand.