Pages 1–6 1cnv Evolutionary trace report by report maker March 21, 2010

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 6

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1cnv): Title: Crystal structure of concanavalin b at 1.65 a resolution Compound: Mol id: 1; molecule: concanavalin b; chain: a Organism, scientific name: Canavalia Ensiformis; 1cnv contains a single unique chain 1cnvA (283 residues long).

CONTENTS 2 CHAIN 1CNVA 1 Introduction 1 2.1 P49347 overview

2 Chain 1cnvA 1 From SwissProt, id P49347, 95% identical to 1cnvA: 2.1 P49347 overview 1 Description: Concanavalin B precursor (Con B). 2.2 Multiple sequence alignment for 1cnvA 1 Organism, scientific name: Canavalia ensiformis (Jack bean) (Horse bean). 2.3 Residue ranking in 1cnvA 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.4 Top ranking residues in 1cnvA and their position on Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core the structure 1 eudicotyledons; rosids; eurosids I; Fabales; Fabaceae; Papilionoi- 2.4.1 Clustering of residues at 25% coverage. 2 deae; Phaseoleae; Canavalia. 2.4.2 Possible novel functional surfaces at 25% Function: May act as a carbohydrate-binding protein. coverage. 2 Similarity: Belongs to the glycosyl hydrolase 18 family. 3 Notes on using trace results 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.1 Coverage 4 collaboration between the Swiss Institute of Bioinformatics and the 3.2 Known substitutions 4 EMBL outstation - the European Bioinformatics Institute. There are 3.3 Surface 4 no restrictions on its use as long as its content is in no way modified 3.4 Number of contacts 4 and this statement is not removed. 3.5 Annotation 4 3.6 Mutation suggestions 4 2.2 Multiple sequence alignment for 1cnvA 4 Appendix 4 For the chain 1cnvA, the alignment 1cnvA.msf (attached) with 152 4.1 File formats 4 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 5 database, and fragments shorter than 75% of the query as well as 4.3 Credits 5 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 5 to this report, under the name of 1cnvA.msf. Its statistics, from the 4.3.2 CE 5 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-141 in 1cnvA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 142-283 in 1cnvA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 3. Residues in 1cnvA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 152 Total number of residues: 40127 Smallest: 221 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 283 Average length: 264.0 Alignment length: 283 Average identity: 46% Most related pair: 99% Most unrelated pair: 19% Most distant seq: 45%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 52% eukaryotic ( 6% fungi, 46% plan- tae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1cnvA.descr. 2.3 Residue ranking in 1cnvA The 1cnvA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1cnvA can be found in the file called 1cnvA.ranks sorted in the attachment. 2.4 Top ranking residues in 1cnvA and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 1cnvA, colored according to the cluster they belong to: residues in the protein . Figure 3 shows residues in 1cnvA colored red, followed by blue and yellow are the largest clusters (see Appendix for by their importance: bright red and yellow indicate more conser- the coloring scheme). Clockwise: front, back, top and bottom views. The ved/important residues (see Appendix for the coloring scheme). A corresponding Pymol script is attached. Pymol script for producing this figure can be found in the attachment. in Table 1. 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the top 25% of all residues, this time colored according to clusters they

2 Table 1. Table 2. continued cluster size member res type substitutions(%) cvg color residues N(1)S(1)E(1)T. red 67 11,12,13,17,19,23,31,35,36 39,40,62,69,72,73,76,79,81 Table 2. Residues forming surface ”patch” in 1cnvA. 82,84,85,90,98,102,105,106 110,112,116,117,118,119,121 123,124,125,127,128,129,130 Table 3. 131,146,159,160,161,162,163 res type disruptive 164,165,168,169,175,176,178 mutations 182,184,185,186,187,188,189 73 Q (Y)(T)(FW)(H) 190,191,225,228,237,265 124 D (R)(H)(FW)(Y) blue 3 259,260,261 123 L (R)(Y)(H)(TK) 106 H (E)(D)(Q)(M) Table 1. Clusters of top ranking residues in 1cnvA. 76 G (R)(K)(E)(Q) 110 L (R)(Y)(H)(T) 112 E (H)(FW)(Y)(R) 2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1cnvA surface, away from (or Table 3. Disruptive mutations for the surface patch in 1cnvA. susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1cnv. It is shown in Fig. 5. The right panel Another group of surface residues is shown in Fig.6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 5. A possible active surface on the chain 1cnvA. The larger cluster it Fig. 6. Another possible active surface on the chain 1cnvA. The larger cluster belongs to is shown in blue. it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table The residues belonging to this surface ”patch” are listed in Table 2, while Table 3 suggests possible disruptive replacements for these 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). residues (see Section 3.6). Table 2. Table 4. res type substitutions(%) cvg res type substitutions(%) cvg antn 73 Q Q(96).K(1)RL 0.04 165 C C(100) 0.00 S-S 124 D D(92)N(5)GAE 0.08 168 P P(96)Q(1)DGFS 0.03 123 L L(71)V(25)I(1)T 0.12 169 D D(95).P(2)QS 0.03 M 189 Y Y(90)F(9) 0.03 106 H W(87)Y(5)F(2) 0.14 164 G Q(75)R(20)LK(1) 0.04 H(1)T(1)Q.(1) AG 76 G G(83)Y(1)DN(11) 0.16 187 R Q(76)R(19)K(2)E 0.05 .AHI N 110 L L(79)G(11)P(1) 0.20 265 W W(94)Y(2)D(1) 0.06 M(3)A(1)F(1). .(1)L 112 E G(88)R(1)V(2)P 0.22 35 F F(95)TY(1)Q.(1) 0.07 continued in next column continued in next column

3 Table 4. continued 3 NOTES ON USING TRACE RESULTS res type substitutions(%) cvg antn 3.1 Coverage 12 G G(96)R.(2) 0.12 131 Q E(74)D(17)A(1) 0.14 Trace results are commonly expressed in terms of coverage: the resi- Q(2)K(1)NL(1)GV due is important if its “coverage” is small - that is if it belongs to 17 G G(92)A(1)P(1)RK 0.15 some small top percentage of residues [100% is all of the residues YQE.(1) in a chain], according to trace. The ET results are presented in the 184 I V(66)I(23)A(9)L 0.17 form of a table, usually limited to top 25% percent of residues (or 190 N N(77)G(10)D(7) 0.17 to some nearby percentage), sorted by the strength of the presumed S(1)YE(1)K evolutionary pressure. (I.e., the smaller the coverage, the stronger the 13 Q Q(73)R(20)TE 0.18 pressure on the residue.) Starting from the top of that list, mutating a N(1)LM.(1) couple of residues should affect the protein somehow, with the exact 11 W W(94)Y(1)G.(2)R 0.19 effects to be determined experimentally. 237 G G(83)A(5)D(3) 0.19 .(3)Y(1)NTV 3.2 Known substitutions 178 T T(82)S(3)Q(3) 0.20 One of the table columns is “substitutions” - other amino acid types N(5).(1)KLE(1)G seen at the same position in the alignment. These amino acid types 176 I L(66)I(27)V(3)M 0.21 may be interchangeable at that position in the protein, so if one wants KF to affect the protein by a point mutation, they should be avoided. For 182 D D(77)T(2)A(3) 0.23 example if the substitutions are “RVK” and the original protein has R(3)H(1)G(2) an R at that position, it is advisable to try anything, but RVK. Conver- S(2)E(5)N sely, when looking for substitutions which will not affect the protein, 133 P G(81)P(4)N(5)FE 0.25 one may try replacing, R with K, or (perhaps more surprisingly), with .D(1)SLK(1)QVA V. The percentage of times the substitution appears in the alignment 191 D N(74)G(1)D(17) 0.25 is given in the immediately following bracket. No percentage is given S(1)E(1)T(1)A 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 Table 4. Residues forming surface ”patch” in 1cnvA. to 100%.

3.3 Surface To detect candidates for novel functional interfaces, first we look for Table 5. residues that are solvent accessible (according to DSSP program) by res type disruptive 2 at least 10A˚ , which is roughly the area needed for one water mole- mutations cule to come in the contact with the residue. Furthermore, we require 165 C (KER)(FQMWHD)(NYLPI)(SVA) that these residues form a “cluster” of residues which have neighbor 168 P (R)(Y)(H)(TK) within 5A˚ from any of their heavy atoms. 169 D (R)(H)(FW)(Y) Note, however, that, if our picture of protein evolution is correct, 189 Y (K)(Q)(EM)(NR) the neighboring residues which are not surface accessible might be 164 G (E)(Y)(HR)(FWD) equally important in maintaining the interaction specificity - they 187 R (T)(Y)(VCAG)(SFWD) should not be automatically dropped from consideration when choo- 265 W (K)(E)(Q)(TR) sing the set for mutagenesis. (Especially if they form a cluster with 35 F (K)(E)(D)(Q) the surface residues.) 12 G (E)(D)(KM)(FW) 131 Q (Y)(H)(FW)(T) 3.4 Number of contacts 17 G (R)(E)(FKWH)(D) 184 I (YR)(H)(TKE)(SQCDG) Another column worth noting is denoted “noc/bb”; it tells the num- 190 N (Y)(FW)(H)(R) ber of contacts heavy atoms of the residue in question make across 13 Q (Y)(H)(FW)(T) the interface, as well as how many of them are realized through the 11 W (E)(K)(D)(Q) backbone atoms (if all or most contacts are through the backbone, 237 G (R)(K)(E)(H) mutation presumably won’t have strong impact). Two heavy atoms 178 T (R)(FWH)(K)(M) are considered to be “in contact” if their centers are closer than 5A˚ . 176 I (Y)(R)(T)(H) 182 D (R)(FW)(H)(K) 3.5 Annotation 133 P (Y)(R)(H)(T) If the residue annotation is available (either from the pdb file or 191 D (R)(H)(FW)(K) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 5. Disruptive mutations for the surface patch in 1cnvA. (indicating existence of related site record in PDB ), S-S (disulfide bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue).

4 3.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following COVERAGE properties: small [AV GSTC], medium [LPNQDEMIK], large [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- V tively [KHR], or negatively [DE] charged, aromatic [WFYH], 100% 50% 30% 5% long aliphatic chain [EKRQM], OH-group possession [SDETY ], and NH2 group possession [NQRK]. The suggestions are listed according to how different they appear to be from the original amino acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid V types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- RELATIVE IMPORTANCE tive to the fold rather than to the interaction. Many researcher will choose, however, the straightforward alanine mutations, especially in the beginning stages of their investigation. Fig. 7. Coloring scheme used to color residues by their relative importance.

4 APPENDIX alignment length (e.g. including gap characters). Also shown are 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- Files with extension “ranks sorted” are the actual trace results. The ned as (idents / MIN(len1, len2)) where idents is the number of fields in the table in this file: exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, • residue# residue number in the PDB file and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best • type amino acid type relative) for all N sequences, then finding the minimum of these N • rank rank of the position according to older version of ET numbers (hence, the most outlying sequence). alistat is copyrighted • variability has two subfields: by HHMI/Washington University School of Medicine, 1992-2001, 1. number of different amino acids appearing in in this column and freely distributed under the GNU General Public License. of the alignment 4.3.2 CE To map ligand binding sites from different 2. their type source structures, report maker uses the CE program: • rho ET score - the smaller this value, the lesser variability of http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) this position across the branches of the tree (and, presumably, ”Protein structure alignment by incremental combinatorial extension the greater the importance for the protein) (CE) of the optimal path . Protein Engineering 11(9) 739-747. • cvg coverage - percentage of the residues on the structure which 4.3.3 DSSP In this work a residue is considered solvent accessi- have this rho or smaller ble if the DSSP program finds it exposed to water by at least 10A˚ 2, • gaps percentage of gaps in this column which is roughly the area needed for one water molecule to come in the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 4.2 Color schemes used Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version The following color scheme is used in figures with residues colored by [email protected] November 18,2002, by cluster size: black is a single-residue cluster; clusters composed of http://www.cmbi.kun.nl/gv/dssp/descrip.html. more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- 4.3.4 HSSP Whenever available, report maker uses HSSP ali- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, gnment as a starting point for the analysis (sequences shorter than bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, 75% of the query are taken out, however); R. Schneider, A. de DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, Daruvar, and C. Sander. ”The HSSP database of protein structure- tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 7. http://swift.cmbi.kun.nl/swift/hssp/ 4.3 Credits 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- 4.3.1 Alistat alistat reads a multiple sequence alignment from the Wesley,” Reading, Mass. (1986). file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4.3.6 Muscle When making alignments “from scratch”, report of residues, the average and range of the sequence lengths, and the maker uses Muscle alignment program: Edgar, Robert C. (2004),

5 ”MUSCLE: multiple sequence alignment with high accuracy and report maker itself is described in Mihalek I., I. Res and O. high throughput.” Nucleic Acids Research 32(5), 1792-97. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics http://www.drive5.com/muscle/ 22:1656-7. 4.3.7 Pymol The figures in this report were produced using 4.6 About report maker Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- report maker was written in 2006 by Ivana Mihalek. The 1D ran- tific LLC (2005). For more information about Pymol see king visualization program was written by Ivica Res.ˇ report maker http://pymol.sourceforge.net/. (Note for Windows is copyrighted by Lichtarge Lab, Baylor College of Medicine, users: the attached package needs to be unzipped for Pymol to read Houston. the scripts and launch the viewer.) 4.7 Attachments 4.4 Note about ET Viewer The following files should accompany this report: Dan Morgan from the Lichtarge lab has developed a visualization • 1cnvA.complex.pdb - coordinates of 1cnvA with all of its tool specifically for viewing trace results. If you are interested, please interacting partners visit: • 1cnvA.etvx - ET viewer input file for 1cnvA http://mammoth.bcm.tmc.edu/traceview/ • 1cnvA.cluster report.summary - Cluster report summary for The viewer is self-unpacking and self-installing. Input files to be used 1cnvA with ETV (extension .etvx) can be found in the attachment to the • 1cnvA.ranks - Ranks file in sequence order for 1cnvA main report. • 1cnvA.clusters - Cluster descriptions for 1cnvA 4.5 Citing this work • 1cnvA.msf - the multiple sequence alignment used for the chain The method used to rank residues and make predictions in this report 1cnvA can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1cnvA.descr - description of sequences used in 1cnvA msf Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1cnvA.ranks sorted - full listing of residues and their ranking by Importance” J. Mol. Bio. 336: 1265-82. For the original version for 1cnvA of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358.

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