Pages 1–8 1jie Evolutionary trace report by report maker December 4, 2009

4.3.1 Alistat 7 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 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 8 4.7 Attachments 8

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1jie): Title: Crystal structure of -binding protein from bleomycin-producing verticillus complexed with CONTENTS metal-free bleomycin Compound: Mol id: 1; molecule: bleomycin-binding protein; chain: 1 Introduction 1 a, b; engineered: yes Organism, scientific name: Streptomyces Verticillus; 2 Chain 1jieA 1 1jie contains a single unique chain 1jieA (122 residues long) and 2.1 Q53793 overview 1 its homologue 1jieB. 2.2 Multiple sequence alignment for 1jieA 1 2.3 Residue ranking in 1jieA 1 2.4 Top ranking residues in 1jieA and their position on the structure 1 2.4.1 Clustering of residues at 25% coverage. 1 2 CHAIN 1JIEA 2.4.2 Overlap with known functional surfaces at 2.1 Q53793 overview 25% coverage. 2 2.4.3 Possible novel functional surfaces at 25% From SwissProt, id Q53793, 100% identical to 1jieA: coverage. 4 Description: Bleomycin/phleomycin binding protein. Organism, scientific name: Streptomyces verticillus. 3 Notes on using trace results 6 Taxonomy: ; ; Actinobacteridae; Actinomy- 3.1 Coverage 6 cetales; Streptomycineae; ; Streptomyces. 3.2 Known substitutions 6 3.3 Surface 6 3.4 Number of contacts 6 3.5 Annotation 7 2.2 Multiple sequence alignment for 1jieA 3.6 Mutation suggestions 7 For the chain 1jieA, the alignment 1jieA.msf (attached) with 42 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 7 database, and fragments shorter than 75% of the query as well as 4.1 File formats 7 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 7 to this report, under the name of 1jieA.msf. Its statistics, from the 4.3 Credits 7 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-122 in 1jieA colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

Format: MSF Number of sequences: 42 Total number of residues: 4574 Smallest: 93 Largest: 122 Average length: 108.9 Alignment length: 122 Average identity: 29% Most related pair: 95% Most unrelated pair: 12% Most distant seq: 32% Fig. 2. Residues in 1jieA, colored by their relative importance. Clockwise: front, back, top and bottom views. Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 16% prokaryotic sequences. (Descripti- ons of some sequences were not readily available.) The file contai- ning the sequence descriptions can be found in the attachment, under the name 1jieA.descr. 2.3 Residue ranking in 1jieA The 1jieA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1jieA can be found in the file called 1jieA.ranks sorted in the attachment. 2.4 Top ranking residues in 1jieA 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 1jieA 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 in Table 1. Fig. 3. Residues in 1jieA, colored according to the cluster they belong to: 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 cluster size member corresponding Pymol script is attached. color residues red 31 9,11,15,19,22,23,27,28,29,33 38,43,44,48,49,50,51,68,70 73,74,104,105,106,110,111 Table 1. continued 112,113,114,116,118 cluster size member continued in next column color residues

Table 1. Clusters of top ranking residues in 1jieA.

2 2.4.2 Overlap with known functional surfaces at 25% coverage. Table 3. continued The name of the ligand is composed of the source PDB identifier res type disruptive and the heteroatom name used in that file. mutations Bleomycin a2 binding site. Table 2 lists the top 25% of residues at the interface with 1jieBLM401 (bleomycin a2). The following table Table 3. List of disruptive mutations for the top 25% of residues in 1jieA, (Table 3) suggests possible disruptive replacements for these residues that are at the interface with bleomycin a2. (see Section 3.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 38 F Y(66) 0.08 32/0 3.37 I(7) W(2) F(19) V(2) E(2) 49 H H(64) 0.12 7/0 3.98 A(4) F(19) R(7) N(2) L(2) 33 F W(38) 0.20 65/0 3.37 F(26) V(2) L(4) Y(7) D(2) A(16) R(2) 51 S S(47) 0.25 9/1 2.56 Fig. 4. Residues in 1jieA, at the interface with bleomycin a2, colored by their Q(7) relative importance. The ligand (bleomycin a2) is colored green. Atoms fur- G(4) ther than 30A˚ away from the geometric center of the ligand, as well as on T(4) the line of sight to the ligand were removed. (See Appendix for the coloring W(7) scheme for the protein chain 1jieA.) L(4) R(7) Figure 4 shows residues in 1jieA colored by their importance, at the F(11) interface with 1jieBLM401. A(2) Bleomycin a2 binding site. Table 4 lists the top 25% of residues at K(2) the interface with 1jieBLM402 (bleomycin a2). The following table (Table 5) suggests possible disruptive replacements for these residues Table 2. The top 25% of residues in 1jieA at the interface with bleomycin (see Section 3.6). a2.(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 Table 4. in the bracket; noc/bb: number of contacts with the ligand, with the number of res type subst’s cvg noc/ dist contacts realized through backbone atoms given in the bracket; dist: distance (%) bb (A˚ ) of closest apporach to the ligand. ) 113 G G(95) 0.03 11/11 2.90 S(2) Y(2) Table 3. 114 N N(76) 0.13 7/7 3.92 res type disruptive F(2) mutations G(2) 38 F (K)(E)(Q)(R) H(7) 49 H (E)(T)(D)(Q) Y(2) 33 F (K)(E)(Q)(TD) V(2) 51 S (KR)(H)(E)(Y) continued in next column continued in next column

3 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) .(2) D(2) S(2)

Table 4. The top 25% of residues in 1jieA at the interface with bleomycin a2.(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 5. res type disruptive mutations 113 G (K)(R)(E)(QM) 114 N (Y)(R)(EH)(FTW)

Table 5. List of disruptive mutations for the top 25% of residues in 1jieA, that are at the interface with bleomycin a2.

Fig. 5. Residues in 1jieA, at the interface with bleomycin a2, colored by their relative importance. The ligand (bleomycin a2) is colored green. Atoms fur- ther than 30A˚ away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1jieA.)

Figure 5 shows residues in 1jieA colored by their importance, at the interface with 1jieBLM402. Interface with 1jieB.Table 6 lists the top 25% of residues at the interface with 1jieB. The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6).

4 Table 6. Table 6. continued res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) 29 F F(97) 0.02 4/0 4.27 68 V V(66) 0.25 31/15 3.36 V(2) L(9) 9 P P(88) 0.04 38/12 3.52 .(2) .(9) G(2) I(2) E(2) 43 R R(83) 0.07 36/8 3.42 C(2) L(9) A(2) H(2) T(4) T(2) M(2) K(2) D(2) 118 F F(73) 0.07 3/0 4.11 I(2) .(21) L(2) Table 6. The top 25% of residues in 1jieA at the interface with 1jieB. I(2) (Field names: res: residue number in the PDB entry; type: amino acid type; 38 F Y(66) 0.08 9/0 3.81 substs: substitutions seen in the alignment; with the percentage of each type I(7) in the bracket; noc/bb: number of contacts with the ligand, with the number of W(2) contacts realized through backbone atoms given in the bracket; dist: distance F(19) of closest apporach to the ligand. ) V(2) E(2) Table 7. 49 H H(64) 0.12 17/0 3.57 A(4) res type disruptive F(19) mutations R(7) 29 F (KE)(QD)(TR)(N) N(2) 9 P (Y)(R)(T)(H) L(2) 43 R (T)(D)(YE)(SCG) 44 G S(19) 0.15 5/5 4.57 118 F (KE)(T)(QDR)(SCG) G(59) 38 F (K)(E)(Q)(R) D(16) 49 H (E)(T)(D)(Q) E(2) 44 G (R)(K)(FWH)(QM) T(2) 70 D (R)(FW)(H)(Y) 70 D D(73) 0.18 7/6 4.03 73 A (R)(K)(YH)(E) N(4) 74 L (Y)(R)(T)(E) S(2) 68 V (R)(Y)(K)(H) G(9) E(4) Table 7. List of disruptive mutations for the top 25% of residues in 1jieA, A(2) that are at the interface with 1jieB. K(2) 73 A A(73) 0.19 4/3 3.96 Figure 6 shows residues in 1jieA colored by their importance, at the E(14) interface with 1jieB. G(2) 2.4.3 Possible novel functional surfaces at 25% coverage. One T(2) group of residues is conserved on the 1jieA surface, away from (or S(7) susbtantially larger than) other functional sites and interfaces reco- 74 L L(59) 0.22 18/2 3.98 gnizable in PDB entry 1jie. It is shown in Fig. 7. The right panel V(19) shows (in blue) the rest of the larger cluster this surface belongs to. F(4) The residues belonging to this surface ”patch” are listed in Table A(7) 8, while Table 9 suggests possible disruptive replacements for these R(2) residues (see Section 3.6). I(7) continued in next column Table 8. res type substitutions(%) cvg 9 P P(88).(9)I(2) 0.04 38 F Y(66)I(7)W(2) 0.08 continued in next column

5 Table 9. res type disruptive mutations 9 P (Y)(R)(T)(H) 38 F (K)(E)(Q)(R) 49 H (E)(T)(D)(Q) 33 F (K)(E)(Q)(TD) 51 S (KR)(H)(E)(Y)

Table 9. Disruptive mutations for the surface patch in 1jieA.

Another group of surface residues is shown in Fig.8. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 6. Residues in 1jieA, at the interface with 1jieB, colored by their relative importance. 1jieB is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1jieA.)

Fig. 8. Another possible active surface on the chain 1jieA. The larger cluster it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 10, while Table 11 suggests possible disruptive replacements for these residues (see Section 3.6). Table 10. res type substitutions(%) cvg 28 G G(100) 0.01 29 F F(97)V(2) 0.02 Fig. 7. A possible active surface on the chain 1jieA. The larger cluster it 27 L L(90)M(2)I(2) 0.03 belongs to is shown in blue. P(2)F(2) 113 G G(95)S(2)Y(2) 0.03 Table 8. continued 110 D D(90)A(2)S(4) 0.05 res type substitutions(%) cvg M(2) F(19)V(2)E(2) 43 R R(83)L(9)H(2) 0.07 49 H H(64)A(4)F(19) 0.12 T(2)K(2) R(7)N(2)L(2) 118 F F(73).(21)L(2) 0.07 33 F W(38)F(26)V(2) 0.20 I(2) L(4)Y(7)D(2) 111 P P(78)L(7)E(9) 0.09 A(16)R(2) I(4) 51 S S(47)Q(7)G(4) 0.25 104 R R(78)K(9)E(2) 0.10 T(4)W(7)L(4) Q(2)P(4)S(2) R(7)F(11)A(2) 15 D S(33)D(59)N(2) 0.12 K(2) E(2).(2) 44 G S(19)G(59)D(16) 0.15 E(2)T(2) Table 8. Residues forming surface ”patch” in 1jieA. continued in next column

6 Table 10. continued 3 NOTES ON USING TRACE RESULTS res type substitutions(%) cvg 3.1 Coverage 22 F F(73)Y(7)H(4) 0.16 W(7)Q(2)R(2) Trace results are commonly expressed in terms of coverage: the resi- L(2) due is important if its “coverage” is small - that is if it belongs to 70 D D(73)N(4)S(2) 0.18 some small top percentage of residues [100% is all of the residues G(9)E(4)A(2) in a chain], according to trace. The ET results are presented in the K(2) form of a table, usually limited to top 25% percent of residues (or 73 A A(73)E(14)G(2) 0.19 to some nearby percentage), sorted by the strength of the presumed T(2)S(7) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 105 E M(7)E(64)D(4) 0.21 pressure on the residue.) Starting from the top of that list, mutating a T(4)V(4)Q(2) couple of residues should affect the protein somehow, with the exact S(4)G(4)R(2) effects to be determined experimentally. 74 L L(59)V(19)F(4) 0.22 A(7)R(2)I(7) 3.2 Known substitutions 19 N A(61)S(14)T(14) 0.23 One of the table columns is “substitutions” - other amino acid types L(4)G(2)N(2) seen at the same position in the alignment. These amino acid types 112 A F(28)D(38)H(4) 0.24 may be interchangeable at that position in the protein, so if one wants S(11)A(9)Y(4) to affect the protein by a point mutation, they should be avoided. For E(2) example if the substitutions are “RVK” and the original protein has 68 V V(66)L(9).(2) 0.25 an R at that position, it is advisable to try anything, but RVK. Conver- G(2)E(2)C(2) sely, when looking for substitutions which will not affect the protein, A(2)T(4)M(2) one may try replacing, R with K, or (perhaps more surprisingly), with D(2)I(2) V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given Table 10. Residues forming surface ”patch” in 1jieA. 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%.

3.3 Surface To detect candidates for novel functional interfaces, first we look for Table 11. residues that are solvent accessible (according to DSSP program) by 2 res type disruptive 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 28 G (KER)(FQMWHD)(NYLPI)(SVA) that these residues form a “cluster” of residues which have neighbor 29 F (KE)(QD)(TR)(N) within 5A˚ from any of their heavy atoms. 27 L (R)(Y)(T)(H) Note, however, that, if our picture of protein evolution is correct, 113 G (K)(R)(E)(QM) the neighboring residues which are not surface accessible might be 110 D (R)(H)(FYW)(K) equally important in maintaining the interaction specificity - they 43 R (T)(D)(YE)(SCG) should not be automatically dropped from consideration when choo- 118 F (KE)(T)(QDR)(SCG) sing the set for mutagenesis. (Especially if they form a cluster with 111 P (YR)(H)(T)(CG) the surface residues.) 104 R (TY)(CDG)(FVAW)(S) 15 D (R)(FWH)(Y)(VCAG) 3.4 Number of contacts 44 G (R)(K)(FWH)(QM) Another column worth noting is denoted “noc/bb”; it tells the num- 22 F (E)(K)(T)(D) ber of contacts heavy atoms of the residue in question make across 70 D (R)(FW)(H)(Y) the interface, as well as how many of them are realized through the 73 A (R)(K)(YH)(E) backbone atoms (if all or most contacts are through the backbone, 105 E (H)(FW)(Y)(R) mutation presumably won’t have strong impact). Two heavy atoms 74 L (Y)(R)(T)(E) are considered to be “in contact” if their centers are closer than 5A˚ . 19 N (Y)(H)(R)(FW) 112 A (K)(R)(E)(Q) 3.5 Annotation 68 V (R)(Y)(K)(H) If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” Table 11. Disruptive mutations for the surface patch in 1jieA. appears. Annotations carried over from PDB are the following: site (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).

7 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. 9. 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. 9. 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),

8 ”MUSCLE: multiple sequence alignment with high accuracy and of service for comparative analysis of proteins.” Bioinformatics high throughput.” Nucleic Acids Research 32(5), 1792-97. 22:1656-7. http://www.drive5.com/muscle/ 4.6 About report maker 4.3.7 Pymol The figures in this report were produced using report maker was written in 2006 by Ivana Mihalek. The 1D ran- Pymol. The scripts can be found in the attachment. Pymol king visualization program was written by Ivica Res.ˇ report maker is an open-source application copyrighted by DeLano Scien- is copyrighted by Lichtarge Lab, Baylor College of Medicine, tific LLC (2005). For more information about Pymol see Houston. http://pymol.sourceforge.net/. (Note for Windows users: the attached package needs to be unzipped for Pymol to read 4.7 Attachments the scripts and launch the viewer.) The following files should accompany this report:

4.4 Note about ET Viewer • 1jieA.complex.pdb - coordinates of 1jieA with all of its interac- Dan Morgan from the Lichtarge lab has developed a visualization ting partners tool specifically for viewing trace results. If you are interested, please • 1jieA.etvx - ET viewer input file for 1jieA visit: • 1jieA.cluster report.summary - Cluster report summary for http://mammoth.bcm.tmc.edu/traceview/ 1jieA The viewer is self-unpacking and self-installing. Input files to be used • 1jieA.ranks - Ranks file in sequence order for 1jieA with ETV (extension .etvx) can be found in the attachment to the • 1jieA.clusters - Cluster descriptions for 1jieA main report. • 1jieA.msf - the multiple sequence alignment used for the chain 4.5 Citing this work 1jieA The method used to rank residues and make predictions in this report • 1jieA.descr - description of sequences used in 1jieA msf can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1jieA.ranks sorted - full listing of residues and their ranking for Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 1jieA by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1jieA.1jieBLM401.if.pml - Pymol script for Figure 4 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1jieA.cbcvg - used by other 1jieA – related pymol scripts tionary Trace Method Defines Binding Surfaces Common to Protein • Families” J. Mol. Bio. 257: 342-358. 1jieA.1jieBLM402.if.pml - Pymol script for Figure 5 report maker itself is described in Mihalek I., I. Res and O. • 1jieA.1jieB.if.pml - Pymol script for Figure 6 Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type

9