Pages 1–12 1w25 Evolutionary trace report by report maker March 6, 2010

4.3.1 Alistat 10 4.3.2 CE 11 4.3.3 DSSP 11 4.3.4 HSSP 11 4.3.5 LaTex 11 4.3.6 Muscle 11 4.3.7 Pymol 11 4.4 Note about ET Viewer 11 4.5 Citing this work 11 4.6 About report maker 11 4.7 Attachments 11

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1w25): Title: Response regulator pled in complex with c-digmp Compound: Mol id: 1; molecule: stalked-cell differentiation control- ling protein; chain: a, b; synonym: diguanylate cyclase; engineered: yes Organism, scientific name: Caulobacter Vibrioides; 1w25 contains a single unique chain 1w25A (454 residues long) and its homologue 1w25B. CONTENTS

1 Introduction 1 2 CHAIN 1W25A 2.1 Q9A5I5 overview 2 Chain 1w25A 1 2.1 Q9A5I5 overview 1 From SwissProt, id Q9A5I5, 97% identical to 1w25A: 2.2 Multiple sequence alignment for 1w25A 1 Description: Response regulator pleD (Stalked-cell differentiation 2.3 Residue ranking in 1w25A 1 controlling protein) [Includes: Diguanylate cyclase (EC 4.6.1.-) 2.4 Top ranking residues in 1w25A and their position on (DGC)]. the structure 2 Organism, scientific name: Caulobacter crescentus. 2.4.1 Clustering of residues at 25% coverage. 2 Taxonomy: Bacteria; Proteobacteria; Alphaproteobacteria; Caulob- 2.4.2 Overlap with known functional surfaces at acterales; Caulobacteraceae; Caulobacter. 25% coverage. 3 Function: Response regulator that is part of a signal transduction 2.4.3 Possible novel functional surfaces at 25% pathway controlling cell differentiation in the swarmer-to-stalked cell coverage. 8 transition. Function: Catalyzes the condensation of two GTP molecules to the 3 Notes on using trace results 10 cyclic dinucleotide di-GMP (c-di-GMP), which acts as a secondary 3.1 Coverage 10 messenger. 3.2 Known substitutions 10 Catalytic activity: 2 GTP = 3’,5’-cyclic di-GMP + 2 diphosphate. 3.3 Surface 10 Enzyme regulation: Allosterically inhibited by the product c-di- 3.4 Number of contacts 10 GMP. 3.5 Annotation 10 Subunit: Homodimer. Inactive monomer in solution. 3.6 Mutation suggestions 10 Subcellular location: Cytoplasmic. Phosphorylated pleD localizes to the differentiating pole. 4 Appendix 10 Domain: PleD is activated by phosphorylation at the first response 4.1 File formats 10 regulatory domain, which induces dimerization mediated by the two 4.2 Color schemes used 10 response regulatory domains and allows the two substrate-binding 4.3 Credits 10 sites to approach each other and the condensation reaction to occur

1 Lichtarge lab 2006 (Probable). The diguanylate cyclase activity is harbored by the GGDEF domain. Ptm: Phosphorylated by pleC and divJ. Phosphorylation stimulates cyclase activity. Similarity: Contains 1 GGDEF domain. Similarity: Contains 2 response regulatory domains. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the 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 1w25A For the chain 1w25A, the alignment 1w25A.msf (attached) with 164 Fig. 1. Residues 2-228 in 1w25A colored by their relative importance. (See sequences was used. The alignment was downloaded from the HSSP Appendix, Fig.14, for the coloring scheme.) 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 1w25A.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 164 Total number of residues: 68534 Smallest: 342 Largest: 454 Average length: 417.9 Alignment length: 454 Average identity: 30% Most related pair: 99% Most unrelated pair: 6% Fig. 2. Residues 229-455 in 1w25A colored by their relative importance. (See Most distant seq: 30% Appendix, Fig.14, for the coloring scheme.)

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 15% 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 1w25A.descr.

2.3 Residue ranking in 1w25A The 1w25A sequence is shown in Figs. 1–2, with each residue colo- red according to its estimated importance. The full listing of residues in 1w25A can be found in the file called 1w25A.ranks sorted in the attachment.

2.4 Top ranking residues in 1w25A 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 1w25A colored by their importance: bright red and yellow indicate more conser- ved/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. 4 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.4 are composed of the residues listed in Table 1.

2 Table 1. continued cluster size member color residues red 73 288,289,292,294,295,296,297 299,300,301,302,303,307,323 324,325,327,328,329,330,331 332,334,335,336,338,339,340 343,344,346,347,351,353,354 359,362,365,366,368,369,370 371,372,373,374,375,376,377 384,388,389,390,391,392,395 412,414,415,416,417,430,434 435,437,438,439,441,442,445 446,447,449 blue 23 5,6,7,8,9,10,14,18,22,25,29 32,35,38,39,47,48,50,51,53 78,105,112 yellow 10 91,94,95,98,99,101,250,260 261,264 green 3 278,279,282 purple 2 244,247 azure 2 271,272 Fig. 3. Residues in 1w25A, colored by their relative importance. Clockwise: front, back, top and bottom views. Table 1. Clusters of top ranking residues in 1w25A.

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. 5GP binding site. Table 2 lists the top 25% of residues at the interface with 1w25B5GP501 (5gp). 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˚ ) 332 K K(96)SH 0.04 5/0 4.62 site QNW

Table 2. The top 25% of residues in 1w25A at the interface with 5GP.(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. )

Fig. 4. Residues in 1w25A, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for Table 3. the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. res type disruptive mutations 332 K (Y)(T)(FW)(CG) Table 1. cluster size member Table 3. List of disruptive mutations for the top 25% of residues in color residues 1w25A, that are at the interface with 5GP. continued in next column Figure 5 shows residues in 1w25A colored by their importance, at the interface with 1w25B5GP501.

3 Fig. 5. Residues in 1w25A, at the interface with 5GP, colored by their relative Fig. 6. Residues in 1w25A, at the interface with 5GP, colored by their relative importance. The ligand (5GP) is colored green. Atoms further than 30A˚ away importance. The ligand (5GP) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line of sight to the 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 ligand were removed. (See Appendix for the coloring scheme for the protein chain 1w25A.) chain 1w25A.)

5GP binding site. Table 4 lists the top 25% of residues at the Figure 6 shows residues in 1w25A colored by their importance, at the interface with 1w25A5GP503 (5gp). The following table (Table 5) interface with 1w25A5GP503. suggests possible disruptive replacements for these residues (see Interface with 1w25B.Table 6 lists the top 25% of residues at Section 3.6). the interface with 1w25B. The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type subst’s cvg noc/ dist antn Table 6. (%) bb (A˚ ) res type subst’s cvg noc/ dist ˚ 359 R R(84). 0.09 14/0 2.84 site (%) bb (A) H(1) 288 L Q(3) 0.19 35/0 1.82 P(3) L(42) G(2)F M(22) E(1)D E(4) S(1)T A(12) K(1)NQ R(2) G(1) .(3) Table 4. The top 25% of residues in 1w25A at the interface with F(1)VWK 5GP.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each YTH(1)C type in the bracket; noc/bb: number of contacts with the ligand, with the num- 25 E A(18) 0.21 19/5 3.22 ber of contacts realized through backbone atoms given in the bracket; dist: E(45) distance of closest apporach to the ligand. ) Q(4) .(16) F(5) Table 5. V(1) res type disruptive P(1) mutations D(3) 359 R (T)(YD)(EVCAG)(S) continued in next column

Table 5. List of disruptive mutations for the top 25% of residues in 1w25A, that are at the interface with 5GP.

4 Table 6. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) K(1) S(1)G T(1) 282 L V(28) 0.22 29/8 1.77 L(30) M(3) .(22) A(3)S Y(3) Q(1)T N(1) F(1)GEH K

Table 6. The top 25% of residues in 1w25A at the interface with 1w25B. (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. ) Fig. 7. Residues in 1w25A, at the interface with 1w25B, colored by their rela- tive importance. 1w25B is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1w25A.) Table 7. res type disruptive mutations Table 8. continued 288 L (YR)(TH)(E)(K) res type subst’s cvg noc/ dist antn 25 E (H)(FW)(R)(Y) (%) bb (A˚ ) 282 L (R)(Y)(H)(T) A(3) E(1)C Table 7. List of disruptive mutations for the top 25% of residues in S(1) 1w25A, that are at the interface with 1w25B. 53 D D(57) 0.19 4/0 2.53 site A(9) N(3) Figure 7 shows residues in 1w25A colored by their importance, at the .(12) interface with 1w25B. F(1)I Magnesium ion binding site. Table 8 lists the top 25% of residues G(1) at the interface with 1w25AMG500 (magnesium ion). The following K(1)T table (Table 9) suggests possible disruptive replacements for these L(1) residues (see Section 3.6). Y(3) Table 8. S(2)C res type subst’s cvg noc/ dist antn V(1)M (%) bb (A˚ ) 105 K K(62) 0.25 1/0 4.86 9 D D(64) 0.13 4/0 2.56 site L(4) .(20) G(6) E(9)AS R(3) G(1) V(1) L(1)RC Q(4) 10 D D(57) 0.17 6/2 2.34 site D(1) T(1) H(1) .(20) A(2)MI N(3) P(1)N H(3) E(1) G(6)P T(3) continued in next column Y(1)S continued in next column

5 Table 8. continued suggests possible disruptive replacements for these residues (see res type subst’s cvg noc/ dist antn Section 3.6). (%) bb (A˚ ) .(1) Table 10. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) Table 8. The top 25% of residues in 1w25A at the interface with magne- sium ion.(Field names: res: residue number in the PDB entry; type: amino 369 G G(99)V 0.00 36/36 2.82 site acid type; substs: substitutions seen in the alignment; with the percentage of 331 F F(98)VP 0.01 14/0 3.80 each type in the bracket; noc/bb: number of contacts with the ligand, with G the number of contacts realized through backbone atoms given in the bracket; 335 N N(97)SF 0.02 16/0 3.13 site dist: distance of closest apporach to the ligand. ) PL 343 G G(98)VA 0.02 5/5 3.67 R Table 9. 344 D D(98)HA 0.02 15/4 2.91 site M res type disruptive 371 E E(97)D 0.02 2/0 4.19 mutations G(1)Q 9 D (R)(H)(FW)(Y) 366 R R(96)P. 0.03 11/0 4.02 10 D (R)(H)(FW)(K) TKG 53 D (R)(H)(FW)(K) 332 K K(96)SH 0.04 15/1 3.53 site 105 K (Y)(FW)(T)(CG) QNW 340 H H(97)DP 0.04 16/5 3.40 site Table 9. List of disruptive mutations for the top 25% of residues in R(1) 1w25A, that are at the interface with magnesium ion. 294 L L(93)IA 0.05 3/0 4.08 M(3)V. 368 G G(96)R. 0.05 3/3 3.90 S(1)DA 370 E E(92) 0.05 25/10 3.51 site D(6)SIR 347 L L(93) 0.06 7/0 3.87 I(3)D Q(1)F

Table 10. The top 25% of residues in 1w25A at the interface with 5GP.(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 11. res type disruptive mutations 369 G (KER)(QHD)(FYMW)(N) 331 F (KE)(QDR)(T)(N) 335 N (Y)(T)(HR)(E) 343 G (E)(K)(DR)(YQH) 344 D (R)(YH)(FKW)(CG) Fig. 8. Residues in 1w25A, at the interface with magnesium ion, colored 371 E (FWH)(YR)(VA)(CG) by their relative importance. The ligand (magnesium ion) is colored green. 366 R (D)(TY)(E)(VLAPI) Atoms further than 30A˚ away from the geometric center of the ligand, as well 332 K (Y)(T)(FW)(CG) as on the line of sight to the ligand were removed. (See Appendix for the 340 H (TE)(QMCDG)(SVA)(K) coloring scheme for the protein chain 1w25A.) 294 L (Y)(R)(H)(T) 368 G (KR)(E)(FWH)(M) 370 E (FWH)(Y)(R)(CG) Figure 8 shows residues in 1w25A colored by their importance, at the continued in next column interface with 1w25AMG500. 5GP binding site. Table 10 lists the top 25% of residues at the interface with 1w25A5GP501 (5gp). The following table (Table 11)

6 Table 11. continued Table 12. continued res type disruptive res type subst’s cvg noc/ dist antn mutations (%) bb (A˚ ) 347 L (R)(Y)(T)(H) H(1) P(3) Table 11. List of disruptive mutations for the top 25% of residues in G(2)F 1w25A, that are at the interface with 5GP. E(1)D S(1)T K(1)NQ 377 P P(71) 0.10 4/4 3.75 T(6)IL. S(5) H(1) R(1) E(1) D(1) A(2)GYV Q(1) N(1)KM 376 M L(46) 0.12 9/2 4.06 M(34) T(1) V(5). I(2)Y F(2)A C(1) Q(3)G 390 R R(72) 0.18 16/0 2.82 site T(3) E(5). A(1) L(1) Fig. 9. Residues in 1w25A, at the interface with 5GP, colored by their relative D(1) importance. The ligand (5GP) is colored green. Atoms further than 30A˚ away N(1) from the geometric center of the ligand, as well as on the line of sight to the S(4) ligand were removed. (See Appendix for the coloring scheme for the protein K(3) chain 1w25A.) Q(2)H

Figure 9 shows residues in 1w25A colored by their importance, at the Table 12. The top 25% of residues in 1w25A at the interface with interface with 1w25A5GP501. 5GP.(Field names: res: residue number in the PDB entry; type: amino acid 5GP binding site. Table 12 lists the top 25% of residues at the type; substs: substitutions seen in the alignment; with the percentage of each interface with 1w25A5GP504 (5gp). The following table (Table 13) type in the bracket; noc/bb: number of contacts with the ligand, with the num- suggests possible disruptive replacements for these residues (see ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Section 3.6). Table 12. res type subst’s cvg noc/ dist antn Table 13. (%) bb (A˚ ) res type disruptive 362 D D(89). 0.08 14/0 2.92 site mutations E(1) 362 D (R)(H)(FW)(KY) M(1) 359 R (T)(YD)(EVCAG)(S) A(1)S 377 P (Y)(R)(H)(T) H(1) 376 M (Y)(H)(R)(T) N(1)F 390 R (T)(Y)(D)(CG) G(1) 359 R R(84). 0.09 71/5 3.34 site Table 13. List of disruptive mutations for the top 25% of residues in continued in next column 1w25A, that are at the interface with 5GP.

7 Table 14. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ )

Table 14. The top 25% of residues in 1w25A at the interface with 5GP.(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 15. res type disruptive mutations 359 R (T)(YD)(EVCAG)(S) 390 R (T)(Y)(D)(CG)

Table 15. List of disruptive mutations for the top 25% of residues in 1w25A, that are at the interface with 5GP.

Fig. 10. Residues in 1w25A, at the interface with 5GP, colored by their rela- tive importance. The ligand (5GP) is colored green. Atoms further 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 1w25A.)

Figure 10 shows residues in 1w25A colored by their importance, at the interface with 1w25A5GP504. 5GP binding site. Table 14 lists the top 25% of residues at the interface with 1w25A5GP506 (5gp). The following table (Table 15) suggests possible disruptive replacements for these residues (see Section 3.6). Table 14. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 359 R R(84). 0.09 50/19 3.00 site H(1) P(3) G(2)F E(1)D S(1)T K(1)NQ 390 R R(72) 0.18 44/0 2.94 site Fig. 11. Residues in 1w25A, at the interface with 5GP, colored by their rela- T(3) tive importance. The ligand (5GP) is colored green. Atoms further than 30A˚ E(5). away from the geometric center of the ligand, as well as on the line of sight A(1) to the ligand were removed. (See Appendix for the coloring scheme for the L(1) protein chain 1w25A.) D(1) N(1) Figure 11 shows residues in 1w25A colored by their importance, at S(4) the interface with 1w25A5GP506. K(3) Q(2)H 2.4.3 Possible novel functional surfaces at 25% coverage. One continued in next column group of residues is conserved on the 1w25A surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1w25. It is shown in Fig. 12. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

8 Table 16. continued res type substitutions(%) cvg antn 78 P P(71)S(1).(7) 0.18 A(2)V(2)L(3) T(1)G(3)R(1) K(4)QDE 53 D D(57)A(9)N(3) 0.19 site .(12)F(1)IG(1) K(1)TL(1)Y(3) S(2)CV(1)M 50 I I(42)V(29).(15) 0.22 D(1)MRA(3)L(3)W Fig. 12. A possible active surface on the chain 1w25A. The larger cluster it Y(1)PTES belongs to is shown in blue. 39 L I(13)L(53)V(1) 0.24 M(2).(16)A(1) R(3)SE(1)G(1) The residues belonging to this surface ”patch” are listed in Table 16, K(3)Q(1)C while Table 17 suggests possible disruptive replacements for these 47 P P(35)C(11)I(9) 0.24 residues (see Section 3.6). .(17)D(2)V(7)H Table 16. S(1)G(3)L(5)WF res type substitutions(%) cvg antn Y(1)KRAN 6 L L(69).(23)M(1)A 0.10 105 K K(62)L(4)G(6) 0.25 I(1)Y(2)FK R(3)V(1)Q(4) 18 L M(3)L(70).(16) 0.12 D(1)H(1)A(2)MI S(1)V(2)DYR(1) P(1)NE(1)T(3) I(1)A(1)H Y(1)S.(1) 8 V V(60).(20)Q(1)R 0.13 MI(3)A(4)DL(3) Table 16. Residues forming surface ”patch” in 1w25A. T(1)GE 9 D D(64).(20)E(9)A 0.13 site SG(1)L(1)RC Table 17. 14 N N(56).(19)F(7) 0.14 res type disruptive S(2)E(1)V(3)I mutations D(1)QM(1)A(1)TH 6 L (YR)(T)(H)(E) RL 18 L (R)(Y)(TH)(K) 32 A A(64)G.(15)S(1) 0.14 8 V (Y)(R)(KH)(E) L(4)C(1)F(2) 9 D (R)(H)(FW)(Y) P(1)DT(1)V(1) 14 N (Y)(H)(T)(FW) I(1)YRE 32 A (R)(K)(E)(Y) 48 D D(58)G(1).(17) 0.16 48 D (R)(H)(FW)(Y) E(3)F(1)S(2) 10 D (R)(H)(FW)(K) A(3)P(1)Q(3) 35 G (R)(E)(K)(H) C(3)H(1)NTVK 38 A (R)(KY)(E)(H) 10 D D(57)T(1).(20) 0.17 site 78 P (Y)(R)(H)(T) N(3)H(3)G(6)P 53 D (R)(H)(FW)(K) A(3)E(1)CS(1) 50 I (R)(Y)(H)(T) 35 G G(54)V(7)A(6) 0.17 39 L (Y)(R)(H)(T) S(4).(17)R(1) 47 P (R)(Y)(T)(H) L(2)KPNM(1)I(1) 105 K (Y)(FW)(T)(CG) Y 38 A A(63)Y.(17)T(1) 0.17 Table 17. Disruptive mutations for the surface patch in 1w25A. F(1)L(3)C(2) I(1)M(1)E(1)N Another group of surface residues is shown in Fig.13. The right panel V(1)GP(1)WK shows (in blue) the rest of the larger cluster this surface belongs to. continued in next column The residues belonging to this surface ”patch” are listed in Table 18, while Table 19 suggests possible disruptive replacements for these residues (see Section 3.6).

9 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to 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 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 Fig. 13. Another possible active surface on the chain 1w25A. The larger effects to be determined experimentally. cluster it belongs to is shown in blue. 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types Table 18. seen at the same position in the alignment. These amino acid types res type substitutions(%) cvg may be interchangeable at that position in the protein, so if one wants 95 L L(67)I(7).(3) 0.15 to affect the protein by a point mutation, they should be avoided. For V(6)PM(4)ET(1)S example if the substitutions are “RVK” and the original protein has F(1)Y(2)RDA(1) an R at that position, it is advisable to try anything, but RVK. Conver- 91 R R(70)C(1)V(2) 0.17 sely, when looking for substitutions which will not affect the protein, .(7)L(3)A(1) one may try replacing, R with K, or (perhaps more surprisingly), with M(1)Y(1)E(1) V. The percentage of times the substitution appears in the alignment I(1)Q(1)S(1) is given in the immediately following bracket. No percentage is given H(1)K(3)DFPW in the cases when it is smaller than 1%. This is meant to be a rough 99 A A(67)SI(7)G(6) 0.17 guide - due to rounding errors these percentages often do not add up V(8).(2)L(1)E to 100%. D(1)M(1)PR(1) 260 E E(67).(7)T(3) 0.18 H(4)KG(3)V(1) 3.3 Surface D(1)RNSQ(3)I(1) To detect candidates for novel functional interfaces, first we look for A(1)FP residues that are solvent accessible (according to DSSP program) by 2 98 G G(74)Y.(3)Q(1) 0.20 at least 10A˚ , which is roughly the area needed for one water mole- L(1)F(1)I(2) cule to come in the contact with the residue. Furthermore, we require W(3)D(1)R(1) that these residues form a “cluster” of residues which have neighbor S(1)T(1)N(1)P within 5A˚ from any of their heavy atoms. V(1)A(1) Note, however, that, if our picture of protein evolution is correct, 261 L M(2)L(64).(6) 0.25 the neighboring residues which are not surface accessible might be V(4)F(7)I(3) equally important in maintaining the interaction specificity - they S(1)TH(1)R(2)K should not be automatically dropped from consideration when choo- Q(1)E(1)A(1)D sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) Table 18. Residues forming surface ”patch” in 1w25A. 3.4 Number of contacts Another column worth noting is denoted “noc/bb”; it tells the num- Table 19. 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 res type disruptive backbone atoms (if all or most contacts are through the backbone, mutations mutation presumably won’t have strong impact). Two heavy atoms 95 L (R)(Y)(H)(T) are considered to be “in contact” if their centers are closer than A˚ . 91 R (T)(D)(Y)(E) 5 99 A (Y)(R)(K)(H) 260 E (H)(FW)(Y)(R) 3.5 Annotation 98 G (R)(K)(E)(H) If the residue annotation is available (either from the pdb file or 261 L (Y)(R)(H)(T) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 19. Disruptive mutations for the surface patch in 1w25A. (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).

10 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. 14. 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. 14. 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),

11 ”MUSCLE: multiple sequence alignment with high accuracy and 4.6 About report maker high throughput.” Nucleic Acids Research 32(5), 1792-97. report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://www.drive5.com/muscle/ king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.7 Pymol The figures in this report were produced using Houston. Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- 4.7 Attachments tific LLC (2005). For more information about Pymol see The following files should accompany this report: http://pymol.sourceforge.net/. (Note for Windows users: the attached package needs to be unzipped for Pymol to read • 1w25A.complex.pdb - coordinates of 1w25A with all of its the scripts and launch the viewer.) interacting partners • 4.4 Note about ET Viewer 1w25A.etvx - ET viewer input file for 1w25A • Dan Morgan from the Lichtarge lab has developed a visualization 1w25A.cluster report.summary - Cluster report summary for tool specifically for viewing trace results. If you are interested, please 1w25A visit: • 1w25A.ranks - Ranks file in sequence order for 1w25A • http://mammoth.bcm.tmc.edu/traceview/ 1w25A.clusters - Cluster descriptions for 1w25A • 1w25A.msf - the multiple sequence alignment used for the chain The viewer is self-unpacking and self-installing. Input files to be used 1w25A with ETV (extension .etvx) can be found in the attachment to the • main report. 1w25A.descr - description of sequences used in 1w25A msf • 1w25A.ranks sorted - full listing of residues and their ranking 4.5 Citing this work for 1w25A The method used to rank residues and make predictions in this report • 1w25A.1w25B5GP501.if.pml - Pymol script for Figure 5 can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1w25A.cbcvg - used by other 1w25A – related pymol scripts Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • by Importance” J. Mol. Bio. 336: 1265-82. For the original version 1w25A.1w25A5GP503.if.pml - Pymol script for Figure 6 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1w25A.1w25B.if.pml - Pymol script for Figure 7 tionary Trace Method Defines Binding Surfaces Common to Protein • 1w25A.1w25AMG500.if.pml - Pymol script for Figure 8 Families” J. Mol. Bio. 257: 342-358. • 1w25A.1w25A5GP501.if.pml - Pymol script for Figure 9 report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1w25A.1w25A5GP504.if.pml - Pymol script for Figure 10 of service for comparative analysis of proteins.” Bioinformatics • 1w25A.1w25A5GP506.if.pml - Pymol script for Figure 11 22:1656-7.

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