Pages 1–7 2bdz Evolutionary trace report by report maker April 16, 2010

4.3.1 Alistat 6 4.3.2 CE 6 4.3.3 DSSP 6 4.3.4 HSSP 6 4.3.5 LaTex 6 4.3.6 Muscle 6 4.3.7 Pymol 6 4.4 Note about ET Viewer 6 4.5 Citing this work 6 4.6 About report maker 6 4.7 Attachments 6

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2bdz): Title: Mexicain from mexicana Compound: Mol id: 1; molecule: mexicain; chain: a, b, c, d; ec: 3.4.22.- Organism, scientific name: Jacaratia Mexicana; 2bdz contains a single unique chain 2bdzA (212 residues long) and its homologues 2bdzD, 2bdzC, and 2bdzB.

CONTENTS 2 CHAIN 2BDZA 2.1 P84346 overview 1 Introduction 1 From SwissProt, id P84346, 99% identical to 2bdzA: 2 Chain 2bdzA 1 Description: Mexicain (EC 3.4.22.-). 2.1 P84346 overview 1 Organism, scientific name: Jacaratia mexicana (Wild papaya) 2.2 Multiple sequence alignment for 2bdzA 1 (Pileus mexicanus). 2.3 Residue ranking in 2bdzA 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.4 Top ranking residues in 2bdzA and their position on Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core the structure 2 eudicotyledons; ; eurosids II; ; ; Jacara- 2.4.1 Clustering of residues at 25% coverage. 2 tia. 2.4.2 Overlap with known functional surfaces at Function: Cysteine protease. 25% coverage. 2 Subcellular location: Secreted. 2.4.3 Possible novel functional surfaces at 25% Tissue specificity: Expressed in latex. coverage. 3 Similarity: Belongs to the peptidase C1 family. About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 5 collaboration between the Swiss Institute of Bioinformatics and the 3.1 Coverage 5 EMBL outstation - the European Bioinformatics Institute. There are 3.2 Known substitutions 5 no restrictions on its use as long as its content is in no way modified 3.3 Surface 5 and this statement is not removed. 3.4 Number of contacts 5 3.5 Annotation 5 2.2 Multiple sequence alignment for 2bdzA 3.6 Mutation suggestions 5 For the chain 2bdzA, the alignment 2bdzA.msf (attached) with 1129 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 5 database, and fragments shorter than 75% of the query as well as 4.1 File formats 5 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 5 to this report, under the name of 2bdzA.msf. Its statistics, from the 4.3 Credits 6 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-106 in 2bdzA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 107-212 in 2bdzA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) Fig. 3. Residues in 2bdzA, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 1129 Total number of residues: 226142 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 123 top 25% of all residues, this time colored according to clusters they Largest: 212 belong to. The clusters in Fig.4 are composed of the residues listed Average length: 200.3 Alignment length: 212 Average identity: 43% Most related pair: 99% Most unrelated pair: 14% Most distant seq: 38%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 48% eukaryotic ( 7% vertebrata, 6% arthropoda, 17% plantae), and 2% viral sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 2bdzA.descr.

2.3 Residue ranking in 2bdzA The 2bdzA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 2bdzA can be found in the file called 2bdzA.ranks sorted in the attachment.

2.4 Top ranking residues in 2bdzA and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 2bdzA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for residues in the protein . Figure 3 shows residues in 2bdzA colored the coloring scheme). Clockwise: front, back, top and bottom views. The by their importance: bright red and yellow indicate more conser- corresponding Pymol script is attached. ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1.

2 Table 1. cluster size member color residues red 42 17,19,22,23,25,26,27,28,29 35,48,49,50,51,52,53,55,56 62,63,65,66,71,74,79,86,87 88,95,141,144,147,159,161 174,175,176,177,178,181,182 185 blue 10 6,7,8,129,164,165,166,167 170,171

Table 1. Clusters of top ranking residues in 2bdzA.

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. E64 binding site. Table 2 lists the top 25% of residues at the inter- face with 2bdzAE64501 (e64). 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˚ ) 22 C C(97) 0.01 1/1 4.59 S-S .(1)PYA FG 66 G G(97) 0.03 34/34 2.65 A(1).CS TERD 19 Q Q(97) 0.04 9/0 2.92 .(1)NHL GPCXEM 159 H H(97) 0.04 28/13 3.29 .(2)TAG FDP 26 W W(91) 0.07 9/3 3.49 Y(5) .(1)SGF APDTCL 25 C C(94) 0.09 28/10 2.33 S(2) .(1)V G(1)DYQ LT 65 G G(94)L. 0.10 30/30 3.07 WQNRTKC AYSDEF 23 G G(91)W 0.19 18/18 3.08 .(1) N(1) S(1)L A(1)YKR continued in next column

3 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) EHMITDV C 158 D N(46) 0.25 29/29 3.17 T(2) D(45)Y .(2) S(1)AHR GIVFL

Table 2. The top 25% of residues in 2bdzA at the interface with E64.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 3. res type disruptive mutations Fig. 5. Residues in 2bdzA, at the interface with E64, colored by their relative 22 C (K)(R)(E)(Q) importance. The ligand (E64) is colored green. Atoms further than 30A˚ away 66 G (R)(K)(FWH)(E) from the geometric center of the ligand, as well as on the line of sight to the 19 Q (Y)(H)(FTW)(S) ligand were removed. (See Appendix for the coloring scheme for the protein 159 H (E)(Q)(K)(MD) chain 2bdzA.) 26 W (K)(E)(Q)(R) 25 C (R)(K)(E)(H) 65 G (R)(K)(E)(H) 23 G (R)(K)(E)(H) 158 D (R)(H)(K)(FW)

Table 3. List of disruptive mutations for the top 25% of residues in 2bdzA, that are at the interface with E64.

Figure 5 shows residues in 2bdzA colored by their importance, at the interface with 2bdzAE64501. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2bdzA surface, away from (or Fig. 6. A possible active surface on the chain 2bdzA. The larger cluster it susbtantially larger than) other functional sites and interfaces reco- belongs to is shown in blue. gnizable in PDB entry 2bdz. It is shown in Fig. 6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Table 4. continued The residues belonging to this surface ”patch” are listed in Table res type substitutions(%) cvg antn 4, while Table 5 suggests possible disruptive replacements for these 19 Q Q(97).(1)NHLGPC 0.04 residues (see Section 3.6). XEM Table 4. 159 H H(97).(2)TAGFDP 0.04 res type substitutions(%) cvg antn 175 N N(97).(2)KHTSGX 0.05 22 C C(97).(1)PYAFG 0.01 S-S 63 C C(97)K.RHGSDNEL 0.06 S-S 88 Y Y(95)F(2)GHMSVR 0.01 YIV DQ 147 G G(96)CV.(1)QDTE 0.06 137 R 145.98 0.01 KARPI 144 Y Y(97)F.PRLV 0.02 26 W W(91)Y(5).(1)SG 0.07 66 G G(97)A(1).CSTER 0.03 FAPDTCL D 35 E E(96).(1)RQSVMG 0.07 continued in next column DKAT continued in next column

4 Table 4. continued Table 5. continued res type substitutions(%) cvg antn res type disruptive 25 C C(94)S(2).(1)V 0.09 mutations G(1)DYQLT 159 H (E)(Q)(K)(MD) 28 F F(96)Q.(1)LRIVW 0.09 175 N (Y)(FW)(H)(E) SPHMT 63 C (R)(KE)(H)(FW) 177 W W(94)V.(2)MY(1) 0.09 147 G (R)(H)(KE)(FW) LHSKFXREC 26 W (K)(E)(Q)(R) 65 G G(94)L.WQNRTKCA 0.10 35 E (H)(FW)(Y)(R) YSDEF 25 C (R)(K)(E)(H) 176 S S(94)T(1)G.(2)W 0.11 28 F (E)(K)(T)(D) KHFEQRIX 177 W (E)(K)(TD)(Q) 182 G G(96)I.(3)DAKEX 0.12 65 G (R)(K)(E)(H) RL 176 S (R)(K)(FWH)(M) 7 W W(93).(3)Y(1)LI 0.13 182 G (R)(H)(KE)(FW) SFRH 7 W (E)(K)(Q)(TD) 87 P P(90)G(1)K(1) 0.14 87 P (Y)(R)(H)(T) S(1)ETRA(1)QVIH 181 W (KE)(Q)(D)(R) YL 6 D (R)(H)(FW)(Y) 181 W W(91)I.(3)F(3) 0.14 50 E (H)(FW)(Y)(R) Y(1)CVSQGXH 161 V (Y)(R)(KE)(H) 6 D D(93).(3)N(2)EA 0.15 164 V (R)(KY)(E)(H) QG 23 G (R)(K)(E)(H) 50 E E(83)Q(2)K(1) 0.16 174 K (Y)(FW)(T)(VCAHG) P(4)V(3)T(1)L 8 R (TD)(Y)(E)(CG) A(1)SYGI.RDM 17 K (Y)(T)(FW)(CG) 161 V V(89)L(1)I(3) 0.16 158 D (R)(H)(K)(FW) M(2).(1)APDR 164 V V(88)I(6)AET(1) 0.18 Table 5. Disruptive mutations for the surface patch in 2bdzA. .(1)GFML 23 G G(91)W.(1)N(1) 0.19 S(1)LA(1)YKREHM ITDVC 3 NOTES ON USING TRACE RESULTS 174 K K(80)ER(15).(2) 0.20 3.1 Coverage LQPGTXN Trace results are commonly expressed in terms of coverage: the resi- 8 R R(89)T(2).(3)KQ 0.21 due is important if its “coverage” is small - that is if it belongs to SV(1)AIHDW some small top percentage of residues [100% is all of the residues 17 K K(85)E(1).(2) 0.22 in a chain], according to trace. The ET results are presented in the R(6)Q(2)IGLYHWN form of a table, usually limited to top 25% percent of residues (or VD to some nearby percentage), sorted by the strength of the presumed 158 D N(46)T(2)D(45)Y 0.25 evolutionary pressure. (I.e., the smaller the coverage, the stronger the .(2)S(1)AHRGIVF pressure on the residue.) Starting from the top of that list, mutating a L couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. Table 4. Residues forming surface ”patch” in 2bdzA. 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types Table 5. seen at the same position in the alignment. These amino acid types res type disruptive may be interchangeable at that position in the protein, so if one wants mutations to affect the protein by a point mutation, they should be avoided. For 22 C (K)(R)(E)(Q) example if the substitutions are “RVK” and the original protein has 88 Y (K)(Q)(M)(E) an R at that position, it is advisable to try anything, but RVK. Conver- 137 R (TD)(YE)(SVCLAPIG)(FMW) sely, when looking for substitutions which will not affect the protein, 144 Y (K)(Q)(E)(M) one may try replacing, R with K, or (perhaps more surprisingly), with 66 G (R)(K)(FWH)(E) V. The percentage of times the substitution appears in the alignment 19 Q (Y)(H)(FTW)(S) is given in the immediately following bracket. No percentage is given continued in next column 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%.

5 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. 7. 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). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 3.6 Mutation suggestions • gaps percentage of gaps in this column Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein The following color scheme is used in figures with residues colored with its ligand. The attempt is made to complement the following by cluster size: black is a single-residue cluster; clusters composed of properties: small [AV GSTC], medium [LPNQDEMIK], large more than one residue colored according to this hierarchy (ordered [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- by descending size): red, blue, yellow, green, purple, azure, tur- tively [KHR], or negatively [DE] charged, aromatic [WFYH], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, long aliphatic chain [EKRQM], OH-group possession [SDETY ], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, and NH2 group possession [NQRK]. The suggestions are listed DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, according to how different they appear to be from the original amino tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. acid, and they are grouped in round brackets if they appear equally The colors used to distinguish the residues by the estimated disruptive. From left to right, each bracketed group of amino acid evolutionary pressure they experience can be seen in Fig. 7. types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will 4.3.1 Alistat alistat reads a multiple sequence alignment from the choose, however, the straightforward alanine mutations, especially in file and shows a number of simple statistics about it. These stati- the beginning stages of their investigation. stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 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- ned as (idents / MIN(len1, len2)) where idents is the number of Files with extension “ranks sorted” are the actual trace results. The exact identities and len1, len2 are the unaligned lengths of the two fields in the table in this file: 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, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • residue# residue number in the PDB file 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

6 by HHMI/Washington University School of Medicine, 1992-2001, http://mammoth.bcm.tmc.edu/traceview/ and freely distributed under the GNU General Public License. The viewer is self-unpacking and self-installing. Input files to be used 4.3.2 CE To map ligand binding sites from different with ETV (extension .etvx) can be found in the attachment to the source structures, report maker uses the CE program: main report. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension 4.5 Citing this work (CE) of the optimal path . Protein Engineering 11(9) 739-747. The method used to rank residues and make predictions in this report 4.3.3 DSSP In this work a residue is considered solvent accessi- can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues which is roughly the area needed for one water molecule to come in by Importance” J. Mol. Bio. 336: 1265-82. For the original version the contact with the residue. DSSP is copyrighted by W. Kabsch, C. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version tionary Trace Method Defines Binding Surfaces Common to Protein by [email protected] November 18,2002, Families” J. Mol. Bio. 257: 342-358. report maker itself is described in Mihalek I., I. Res and O. http://www.cmbi.kun.nl/gv/dssp/descrip.html. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 22:1656-7. gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de 4.6 About report maker Daruvar, and C. Sander. ”The HSSP database of protein structure- report maker was written in 2006 by Ivana Mihalek. The 1D ran- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. king visualization program was written by Ivica Res.ˇ report maker http://swift.cmbi.kun.nl/swift/hssp/ is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- 4.7 Attachments Wesley,” Reading, Mass. (1986). The following files should accompany this report: 4.3.6 Muscle When making alignments “from scratch”, report • 2bdzA.complex.pdb - coordinates of 2bdzA with all of its maker uses Muscle alignment program: Edgar, Robert C. (2004), interacting partners ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. • 2bdzA.etvx - ET viewer input file for 2bdzA • http://www.drive5.com/muscle/ 2bdzA.cluster report.summary - Cluster report summary for 2bdzA 4.3.7 Pymol The figures in this report were produced using • 2bdzA.ranks - Ranks file in sequence order for 2bdzA Pymol. The scripts can be found in the attachment. Pymol • 2bdzA.clusters - Cluster descriptions for 2bdzA is an open-source application copyrighted by DeLano Scien- tific LLC (2005). For more information about Pymol see • 2bdzA.msf - the multiple sequence alignment used for the chain http://pymol.sourceforge.net/. (Note for Windows 2bdzA users: the attached package needs to be unzipped for Pymol to read • 2bdzA.descr - description of sequences used in 2bdzA msf the scripts and launch the viewer.) • 2bdzA.ranks sorted - full listing of residues and their ranking 4.4 Note about ET Viewer for 2bdzA Dan Morgan from the Lichtarge lab has developed a visualization • 2bdzA.2bdzAE64501.if.pml - Pymol script for Figure 5 tool specifically for viewing trace results. If you are interested, please • 2bdzA.cbcvg - used by other 2bdzA – related pymol scripts visit:

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