Pages 1–7 1e5o Evolutionary trace report by report maker September 20, 2008

4.3.1 Alistat 6 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 4.3.7 Pymol 7 4.4 Note about ET Viewer 7 4.5 Citing this work 7 4.6 About report maker 7 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1e5o): Title: Endothiapepsin complex with inhibitor db2 Compound: Mol id: 1; molecule: endothiapepsin; chain: e; frag- ment: residue 90-419; ec: 3.4.23.23; mol id: 2; molecule: endothia- pepsin inhibitor; chain: i Organism, scientific name: Endothia Parasitica; 1e5o contains a single unique chain 1e5oE (330 residues long). Chain 1e5oI is too short (4 residues) to permit statistically significant CONTENTS analysis, and was treated as a peptide ligand.

1 Introduction 1 2 CHAIN 1E5OE 2.1 P11838 overview 2 Chain 1e5oE 1 2.1 P11838 overview 1 From SwissProt, id P11838, 90% identical to 1e5oE: 2.2 Multiple sequence alignment for 1e5oE 1 Description: Endothiapepsin precursor (EC 3.4.23.22) (Aspartate 2.3 Residue ranking in 1e5oE 1 protease). 2.4 Top ranking residues in 1e5oE and their position on Organism, scientific name: parasitica (Chesnut the structure 2 blight ) (Endothia parasitica). 2.4.1 Clustering of residues at 25% coverage. 2 Taxonomy: Eukaryota; Fungi; ; ; 2.4.2 Overlap with known functional surfaces at ; ; ; Valsaceae; 25% coverage. 3 Cryphonectria-Endothia complex; Cryphonectria. 2.4.3 Possible novel functional surfaces at 25% Catalytic activity: Hydrolysis of proteins with specificity similar to coverage. 3 that of pepsin A, prefers hydrophobic residues at P1 and P1’, but does not cleave 14-Ala-—-Leu-15 in the B chain of insulin or Z-Glu-Tyr. 3 Notes on using trace results 5 Clots milk. 3.1 Coverage 5 Similarity: Belongs to the peptidase A1 family. 3.2 Known substitutions 5 About: This Swiss-Prot entry is copyright. It is produced through a 3.3 Surface 6 collaboration between the Swiss Institute of Bioinformatics and the 3.4 Number of contacts 6 EMBL outstation - the European Bioinformatics Institute. There are 3.5 Annotation 6 no restrictions on its use as long as its content is in no way modified 3.6 Mutation suggestions 6 and this statement is not removed.

4 Appendix 6 2.2 Multiple sequence alignment for 1e5oE 4.1 File formats 6 For the chain 1e5oE, the alignment 1e5oE.msf (attached) with 114 4.2 Color schemes used 6 sequences was used. The alignment was assembled through combi- 4.3 Credits 6 nation of BLAST searching on the UniProt database and alignment

1 Lichtarge lab 2006 2.4 Top ranking residues in 1e5oE and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1e5oE 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.

Fig. 1. Residues -2-159 in 1e5oE colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.) Note that some residues in 1e5oE carry insertion code.

Fig. 2. Residues 162-326 in 1e5oE colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.) Note that some residues in 1e5oE carry insertion code.

using Muscle program. It can be found in the attachment to this Fig. 3. Residues in 1e5oE, colored by their relative importance. Clockwise: alistat report, under the name of 1e5oE.msf. Its statistics, from the front, back, top and bottom views. program are the following:

Format: MSF Number of sequences: 114 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 35559 top 25% of all residues, this time colored according to clusters they Smallest: 270 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 330 in Table 1. Average length: 311.9 Alignment length: 330 Table 1. Average identity: 36% cluster size member Most related pair: 98% color residues Most unrelated pair: 17% red 82 11,14,18,21,23,25,29,31,32 Most distant seq: 32% 33,34,35,36,37,38,39,40,42 45,56,61,63,71,73,75,76,77 78,80,82,87,99,101,102,104 Furthermore, 1% of residues show as conserved in this alignment. 108,111,118,119,120,121,122 The alignment consists of 99% eukaryotic ( 34% vertebrata, 5% 123,137,146,151,153,155,167 arthropoda, 43% fungi) sequences. (Descriptions of some sequences 168,171,175,177,190,213,214 were not readily available.) The file containing the sequence descrip- 215,216,217,218,219,220,224 tions can be found in the attachment, under the name 1e5oE.descr. 250,255,273,283,285,286,300 301,303,304,306,307,308,311 2.3 Residue ranking in 1e5oE 313,314,315,322,324 The 1e5oE sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues Table 1. Clusters of top ranking residues in 1e5oE. in 1e5oE can be found in the file called 1e5oE.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) V(7) L(9)TMK 76 G G(91) 0.13 19/19 2.79 E(2) V(1)D.I PA 213 I I(84) 0.13 4/0 4.04 L(3) V(6) A(3) T(2) 111 F F(71) 0.14 8/0 3.98 .(6) L(12) Y(1) M(3) E(1)VTQ A 77 D S(24) 0.21 11/0 2.97 Fig. 4. Residues in 1e5oE, colored according to the cluster they belong to: D(35) red, followed by blue and yellow are the largest clusters (see Appendix for T(36)K. the coloring scheme). Clockwise: front, back, top and bottom views. The LG corresponding Pymol script is attached. Table 2. The top 25% of residues in 1e5oE at the interface with 1e5oI. (Field names: res: residue number in the PDB entry; type: amino acid type; 2.4.2 Overlap with known functional surfaces at 25% coverage. substs: substitutions seen in the alignment; with the percentage of each type The name of the ligand is composed of the source PDB identifier in the bracket; noc/bb: number of contacts with the ligand, with the number of and the heteroatom name used in that file. contacts realized through backbone atoms given in the bracket; dist: distance Interface with the peptide 1e5oI. Table 2 lists the top 25% of of closest apporach to the ligand. ) residues at the interface with 1e5oI. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. Table 3. res type subst’s cvg noc/ dist antn res type disruptive (%) bb (A˚ ) mutations 32 D D(100) 0.01 21/0 2.85 site 32 D (R)(FWH)(KYVCAG)(TQM) 215 D D(100) 0.01 19/0 2.95 site 215 D (R)(FWH)(KYVCAG)(TQM) 35 S S(98) 0.02 11/7 3.70 35 S (KR)(FQMWH)(E)(NYLPI) G(1) 217 G (KR)(E)(FQMWH)(D) 217 G G(99)S 0.02 20/20 3.19 34 G (KR)(E)(QH)(D) 34 G G(97)VA 0.04 25/25 3.04 75 Y (KM)(EVQLAPI)(ND)(SCRG) T 218 T (KR)(FQMWH)(NELPI)(D) 75 Y Y(98) 0.04 74/26 3.13 120 L (R)(Y)(H)(K) R(1) 301 I (R)(Y)(H)(K) 218 T T(97) 0.04 12/4 3.88 73 I (Y)(R)(H)(T) S(2) 76 G (R)(K)(H)(E) 120 L I(71) 0.06 9/0 3.70 213 I (R)(Y)(H)(K) L(27)T 111 F (K)(E)(R)(TQ) 301 I I(90) 0.10 4/0 3.70 77 D (R)(FWH)(Y)(K) V(3) L(5)T Table 3. List of disruptive mutations for the top 25% of residues in 73 I I(80) 0.12 3/0 4.35 1e5oE, that are at the interface with 1e5oI. continued in next column

Figure 5 shows residues in 1e5oE colored by their importance, at the interface with 1e5oI.

3 Table 4. continued res type substitutions(%) cvg .(5)Q(1)S(2) 25 Q Q(83)T.(6)V(4) 0.18 K(1)RDEI 63 T T(86)S(11)KQ 0.22

Table 4. Residues forming surface ”patch” in 1e5oE.

Table 5. res type disruptive mutations 21 G (KER)(FQMWHD)(NLPI)(Y) 61 S (R)(K)(H)(FW) 23 P (Y)(R)(H)(T) 25 Q (Y)(FWH)(T)(CG) 63 T (FW)(HR)(K)(M)

Table 5. Disruptive mutations for the surface patch in 1e5oE.

Another group of surface residues is shown in Fig.7. The right panel Fig. 5. Residues in 1e5oE, at the interface with 1e5oI, colored by their rela- tive importance. 1e5oI is shown in backbone representation (See Appendix shows (in blue) the rest of the larger cluster this surface belongs to. for the coloring scheme for the protein chain 1e5oE.)

2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1e5oE surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1e5o. It is shown in Fig. 6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 7. Another possible active surface on the chain 1e5oE. The larger cluster it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 6, while Table 7 suggests possible disruptive replacements for these residues (see Section 3.6). Table 6. Fig. 6. A possible active surface on the chain 1e5oE. The larger cluster it res type substitutions(%) cvg antn belongs to is shown in blue. 32 D D(100) 0.01 site 82 G G(100) 0.01 122 G G(100) 0.01 The residues belonging to this surface ”patch” are listed in Table 215 D D(100) 0.01 site 4, while Table 5 suggests possible disruptive replacements for these 35 S S(98)G(1) 0.02 residues (see Section 3.6). 217 G G(99)S 0.02 Table 4. 34 G G(97)VAT 0.04 res type substitutions(%) cvg 75 Y Y(98)R(1) 0.04 21 G G(99). 0.03 218 T T(97)S(2) 0.04 61 S S(95)N(1)T(1)L 0.06 216 T T(90)S(8)F 0.05 23 P P(82)N(5)T(2) 0.18 continued in next column continued in next column

4 Table 6. continued Table 7. res type substitutions(%) cvg antn res type disruptive 120 L I(71)L(27)T 0.06 mutations 308 K R(45)G(18)K(34) 0.07 32 D (R)(FWH)(KYVCAG)(TQM) Q(1) 82 G (KER)(FQMWHD)(NYLPI)(SVA) 14 Y Y(92)W(5).FC 0.08 122 G (KER)(FQMWHD)(NYLPI)(SVA) 78 G G(95)K(1)Q.S 0.08 215 D (R)(FWH)(KYVCAG)(TQM) 304 D D(92)ESGVN(1)AT 0.08 35 S (KR)(FQMWH)(E)(NYLPI) 190 W Y(9)L(4)W(85)F 0.09 217 G (KR)(E)(FQMWH)(D) 99 Q T(6)Q(85)A(1)F 0.10 34 G (KR)(E)(QH)(D) M(1)L(1)Y(1) 75 Y (KM)(EVQLAPI)(ND)(SCRG) 123 L L(74)I(6)M(17)S 0.10 218 T (KR)(FQMWH)(NELPI)(D) A 216 T (K)(R)(Q)(M) 301 I I(90)V(3)L(5)T 0.10 120 L (R)(Y)(H)(K) 307 L I(49)L(46)F(3)M 0.11 308 K (Y)(FW)(T)(SVA) 73 I I(80)V(7)L(9)TM 0.12 14 Y (K)(Q)(M)(E) K 78 G (FW)(R)(EH)(K) 219 T S(62)T(35)P(1)N 0.12 304 D (R)(H)(FW)(K) 76 G G(91)E(2)V(1)D. 0.13 190 W (K)(E)(Q)(TD) IPA 99 Q (Y)(H)(T)(FW) 213 I I(84)L(3)V(6) 0.13 123 L (YR)(H)(T)(K) A(3)T(2) 301 I (R)(Y)(H)(K) 38 L L(86)F(2)I(1) 0.14 307 L (YR)(T)(H)(SKECG) M(1)T(5)V(1) 73 I (Y)(R)(H)(T) 111 F F(71).(6)L(12) 0.14 219 T (R)(K)(H)(FW) Y(1)M(3)E(1)VTQ 76 G (R)(K)(H)(E) A 213 I (R)(Y)(H)(K) 220 L F(5)L(78)V(1) 0.16 38 L (R)(Y)(H)(TK) W(3)M(2)I(3) 111 F (K)(E)(R)(TQ) Y(2)QK 220 L (Y)(R)(T)(H) 118 D D(78).(6)N(2)QG 0.17 118 D (R)(H)(FW)(K) AE(3)YS(5)T 224 P (Y)(R)(H)(T) 224 P P(83)S(4)D(9) 0.18 36 S (R)(K)(H)(Y) Q(1)N 71 W (K)(E)(R)(TD) 36 S S(80)A(18)P 0.20 77 D (R)(FWH)(Y)(K) 71 W W(32)F(43)V(4) 0.20 102 E (H)(FW)(R)(Y) L(11)M(2)I(4)T 300 N (Y)(H)(R)(FW) 77 D S(24)D(35)T(36) 0.21 137 T (R)(K)(H)(FW) K.LG 108 S (KR)(Q)(FMWH)(E) 102 E G(54)A(17)S 0.21 153 A (KE)(R)(Y)(D) E(22)Q(2)TL 315 N (Y)(FW)(H)(T) 300 N W(61)T(5)V(1) 0.21 S(8)N(7)A(7)E Table 7. Disruptive mutations for the surface patch in 1e5oE. D(2)Q(1)MY 137 T P(58)T(35)S(3). 0.23 AN 108 S S(38)P(40)A(14) 0.24 3 NOTES ON USING TRACE RESULTS .(1)T(1)HGDR 153 A V(50)L(6)F(28) 0.24 3.1 Coverage I(2)A(8)RTMY Trace results are commonly expressed in terms of coverage: the resi- 315 N D(76)N(16)KAST 0.25 due is important if its “coverage” is small - that is if it belongs to .(1)RE some small top percentage of residues [100% is all of the residues in a chain], according to trace. The ET results are presented in the Table 6. Residues forming surface ”patch” in 1e5oE. form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the pressure on the residue.) Starting from the top of that list, mutating a couple of residues should affect the protein somehow, with the exact effects to be determined experimentally.

5 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types may be interchangeable at that position in the protein, so if one wants to affect the protein by a point mutation, they should be avoided. For COVERAGE example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- V sely, when looking for substitutions which will not affect the protein, 100% 50% 30% 5% one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given 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 V to 100%. RELATIVE IMPORTANCE 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by Fig. 8. Coloring scheme used to color residues by their relative importance. 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 that these residues form a “cluster” of residues which have neighbor disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- 4 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with 4.1 File formats the surface residues.) Files with extension “ranks sorted” are the actual trace results. The 3.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • alignment# number of the position in the alignment 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 • residue# residue number in the PDB file backbone atoms (if all or most contacts are through the backbone, • type amino acid type mutation presumably won’t have strong impact). Two heavy atoms • rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 3.5 Annotation 1. number of different amino acids appearing in in this column If the residue annotation is available (either from the pdb file or of the alignment from other sources), another column, with the header “annotation” 2. their type appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller Mutation suggestions are completely heuristic and based on comple- • gaps percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GST C], medium [LP NQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [W F Y H], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDET Y ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 8.

6 4.3 Credits http://pymol.sourceforge.net/. (Note for Windows 4.3.1 Alistat alistat reads a multiple sequence alignment from the users: the attached package needs to be unzipped for Pymol to read file and shows a number of simple statistics about it. These stati- the scripts and launch the viewer.) stics include the format, the number of sequences, the total number 4.4 Note about ET Viewer of residues, the average and range of the sequence lengths, and the Dan Morgan from the Lichtarge lab has developed a visualization alignment length (e.g. including gap characters). Also shown are tool specifically for viewing trace results. If you are interested, please some percent identities. A percent pairwise alignment identity is defi- visit: ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two http://mammoth.bcm.tmc.edu/traceview/ sequences. The ”average percent identity”, ”most related pair”, and The viewer is self-unpacking and self-installing. Input files to be used ”most unrelated pair” of the alignment are the average, maximum, with ETV (extension .etvx) can be found in the attachment to the and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant main report. seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N 4.5 Citing this work numbers (hence, the most outlying sequence). alistat is copyrighted The method used to rank residues and make predictions in this report by HHMI/Washington University School of Medicine, 1992-2001, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and freely distributed under the GNU General Public License. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 4.3.2 CE To map ligand binding sites from different by Importance” J. Mol. Bio. 336: 1265-82. For the original version source structures, report maker uses the CE program: of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tionary Trace Method Defines Binding Surfaces Common to Protein ”Protein structure alignment by incremental combinatorial extension Families” J. Mol. Bio. 257: 342-358. (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.3 DSSP In this work a residue is considered solvent accessi- of service for comparative analysis of proteins.” 2 Bioinformatics ble if the DSSP program finds it exposed to water by at least 10A˚ , 22:1656-7. 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. 4.6 About report maker Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version report maker was written in 2006 by Ivana Mihalek. The 1D ran- by [email protected] November 18,2002, king visualization program was written by Ivica Res.ˇ report maker http://www.cmbi.kun.nl/gv/dssp/descrip.html. is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 4.7 Attachments gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de The following files should accompany this report: Daruvar, and C. Sander. ”The HSSP database of protein structure- • 1e5oE.complex.pdb - coordinates of 1e5oE with all of its inter- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. acting partners http://swift.cmbi.kun.nl/swift/hssp/ • 1e5oE.etvx - ET viewer input file for 1e5oE • 4.3.5 LaTex The text for this report was processed using LATEX; 1e5oE.cluster report.summary - Cluster report summary for Leslie Lamport, “LaTeX: A Document Preparation System Addison- 1e5oE Wesley,” Reading, Mass. (1986). • 1e5oE.ranks - Ranks file in sequence order for 1e5oE 4.3.6 Muscle When making alignments “from scratch”, report • 1e5oE.clusters - Cluster descriptions for 1e5oE maker uses Muscle alignment program: Edgar, Robert C. (2004), • 1e5oE.msf - the multiple sequence alignment used for the chain ”MUSCLE: multiple sequence alignment with high accuracy and 1e5oE high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1e5oE.descr - description of sequences used in 1e5oE msf http://www.drive5.com/muscle/ • 1e5oE.ranks sorted - full listing of residues and their ranking for 4.3.7 Pymol The figures in this report were produced using 1e5oE Pymol. The scripts can be found in the attachment. Pymol • 1e5oE.1e5oI.if.pml - Pymol script for Figure 5 is an open-source application copyrighted by DeLano Scien- • 1e5oE.cbcvg - used by other 1e5oE – related pymol scripts tific LLC (2005). For more information about Pymol see

7