Pages 1–7 1ki9 Evolutionary trace report by report maker July 15, 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 7 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1ki9): Title: Adenylate kinase from thermolithotrophicus Compound: Mol id: 1; molecule: adenylate kinase; chain: a, b, c; synonym: atp-amp transphosphorylase; ec: 2.7.4.3; engineered: yes Organism, scientific name: Methanococcus Thermolithotrophicus; 1ki9 contains a single unique chain 1ki9A (191 residues long) and its homologues 1ki9C and 1ki9B.

CONTENTS 2 CHAIN 1KI9A 2.1 P43410 overview 1 Introduction 1 From SwissProt, id P43410, 92% identical to 1ki9A: 2 Chain 1ki9A 1 Description: Adenylate kinase (EC 2.7.4.3) (ATP-AMP transphos- 2.1 P43410 overview 1 phorylase). 2.2 Multiple sequence alignment for 1ki9A 1 Organism, scientific name: Methanococcus thermolithotrophicus. 2.3 Residue ranking in 1ki9A 1 : ; ; ; Methanococca- 2.4 Top ranking residues in 1ki9A and their position on les; Methanococcaceae; . the structure 2 Catalytic activity: ATP + AMP = 2 ADP. 2.4.1 Clustering of residues at 25% coverage. 2 Biophysicochemical properties: 2.4.2 Overlap with known functional surfaces at Temperature dependence: Active from 60 to 80 degrees Celsius; 25% coverage. 2 Subunit: Monomer (Probable). 2.4.3 Possible novel functional surfaces at 25% Subcellular location: Cytoplasmic. coverage. 3 Similarity: Belongs to the archaeal adenylate kinase 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 1ki9A 3.6 Mutation suggestions 5 For the chain 1ki9A, the alignment 1ki9A.msf (attached) with 48 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 6 to this report, under the name of 1ki9A.msf. Its statistics, from the 4.3 Credits 6 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 2-192 in 1ki9A colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Format: MSF Number of sequences: 48 Total number of residues: 8818 Smallest: 147 Largest: 191 Average length: 183.7 Alignment length: 191 Average identity: 43% Most related pair: 98% Fig. 2. Residues in 1ki9A, colored by their relative importance. Clockwise: Most unrelated pair: 25% front, back, top and bottom views. Most distant seq: 36%

Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 4% prokaryotic, and 37% archaean sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 1ki9A.descr. 2.3 Residue ranking in 1ki9A The 1ki9A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1ki9A can be found in the file called 1ki9A.ranks sorted in the attachment. 2.4 Top ranking residues in 1ki9A 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 1ki9A 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 Fig. 3. Residues in 1ki9A, colored according to the cluster they belong to: belong to. The clusters in Fig.3 are composed of the residues listed red, followed by blue and yellow are the largest clusters (see Appendix for in Table 1. the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 1. cluster size member Table 1. continued color residues cluster size member red 44 10,12,13,15,18,38,40,43,47 color residues 56,57,60,62,67,70,71,74,75 103,104,105,106,109,122,128 78,90,91,92,93,95,97,100,102 131,132,135,138,140,141,155 continued in next column 156,162,175 continued in next column

2 Table 1. continued cluster size member color residues

Table 1. Clusters of top ranking residues in 1ki9A.

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. Interface with 1ki9C.Table 2 lists the top 25% of residues at the interface with 1ki9C. 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 (%) bb (A˚ ) 122 E E(87) 0.16 1/0 4.65 T(8) Y(2) S(2) 167 G E(12) 0.17 17/17 3.66 G(70) Fig. 4. Residues in 1ki9A, at the interface with 1ki9C, colored by their rela- N(10) tive importance. 1ki9C is shown in backbone representation (See Appendix A(4) for the coloring scheme for the protein chain 1ki9A.) .(2) 156 R R(85) 0.19 30/6 3.34 D(8) Q(4) S(2) 170 V V(72) 0.23 50/41 2.60 I(14) M(10) .(2)

Table 2. The top 25% of residues in 1ki9A at the interface with 1ki9C. (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 3. res type disruptive mutations 122 E (FWHR)(KVA)(YCG)(NLPI) 167 G (R)(H)(K)(FW) 156 R (TY)(FVCAWDG)(LPI)(SE) 170 V (Y)(R)(KEH)(D)

Table 3. List of disruptive mutations for the top 25% of residues in 1ki9A, that are at the interface with 1ki9C.

Figure 4 shows residues in 1ki9A colored by their importance, at the interface with 1ki9C. Interface with 1ki9B.Table 4 lists the top 25% of residues at the interface with 1ki9B. The following table (Table 5) suggests possible disruptive replacements for these residues (see Section 3.6).

3 Table 4. res type subst’s cvg noc/ dist (%) bb (A˚ ) 100 G G(100) 0.03 25/25 3.71 106 P P(100) 0.03 11/8 3.65 105 L L(95) 0.12 4/4 3.86 I(2) F(2) 103 P P(77) 0.17 38/3 3.35 A(12) L(8) I(2) 97 T I(10) 0.19 1/1 4.75 T(81) S(6) M(2) 102 L Y(25) 0.21 16/7 3.39 L(64) W(8) M(2) 162 Y A(12) 0.21 9/0 3.55 Y(54) C(14) Fig. 5. Residues in 1ki9A, at the interface with 1ki9B, colored by their rela- L(2) tive importance. 1ki9B is shown in backbone representation (See Appendix S(14) for the coloring scheme for the protein chain 1ki9A.) .(2)

Table 4. The top 25% of residues in 1ki9A at the interface with 1ki9B. (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 Fig. 6. A possible active surface on the chain 1ki9A. The larger cluster it 100 G (KER)(FQMWHD)(NYLPI)(SVA) belongs to is shown in blue. 106 P (YR)(TH)(SKECG)(FQWD) 105 L (R)(Y)(T)(KE) 103 P (YR)(H)(T)(KE) 6, while Table 7 suggests possible disruptive replacements for these 97 T (R)(K)(H)(FW) residues (see Section 3.6). 102 L (R)(TY)(K)(EH) Table 6. 162 Y (K)(Q)(R)(M) res type substitutions(%) cvg 43 M M(100) 0.03 Table 5. List of disruptive mutations for the top 25% of residues in 1ki9A, 92 H H(100) 0.03 that are at the interface with 1ki9B. 100 G G(100) 0.03 106 P P(100) 0.03 131 R R(100) 0.03 Figure 5 shows residues in 1ki9A colored by their importance, at the 90 D D(97)T(2) 0.04 interface with 1ki9B. 135 D D(97)K(2) 0.04 2.4.3 Possible novel functional surfaces at 25% coverage. One 140 R R(97)Y(2) 0.04 group of residues is conserved on the 1ki9A surface, away from (or 56 R R(97)L(2) 0.07 susbtantially larger than) other functional sites and interfaces reco- 71 Q Q(97)W(2) 0.07 gnizable in PDB entry 1ki9. It is shown in Fig. 6. The right panel continued in next column shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table

4 Table 6. continued Table 7. continued res type substitutions(%) cvg res type disruptive 91 T T(97)L(2) 0.07 mutations 104 G G(97)S(2) 0.07 131 R (TD)(SYEVCLAPIG)(FMW)(N) 138 R R(97)K(2) 0.07 90 D (R)(FWH)(K)(YVQMA) 57 D D(95)E(2)A(2) 0.08 135 D (FW)(YHR)(VCAG)(T) 60 R R(95)F(2)G(2) 0.08 140 R (D)(TEVLAPI)(SMCG)(FNYW) 132 R R(85)Q(14) 0.08 56 R (T)(YD)(SECG)(VA) 15 G G(97).(2) 0.10 71 Q (TY)(SCHG)(FVAWD)(E) 40 G G(97).(2) 0.10 91 T (R)(K)(H)(FQW) 105 L L(95)I(2)F(2) 0.12 104 G (KR)(E)(FQMWH)(D) 13 G G(95).(4) 0.13 138 R (T)(YD)(SVCAG)(FELWPI) 62 L K(12)L(83)M(4) 0.14 57 D (R)(H)(FW)(Y) 67 Q Q(87)Y(8)T(2) 0.14 60 R (D)(E)(T)(SYLPI) E(2) 132 R (T)(YD)(SVCAG)(FELWPI) 10 G A(12)G(83).(4) 0.15 15 G (KER)(FQMWHD)(NLPI)(Y) 175 N E(2)N(85)F(10) 0.15 40 G (KER)(FQMWHD)(NLPI)(Y) .(2) 105 L (R)(Y)(T)(KE) 122 E E(87)T(8)Y(2) 0.16 13 G (KER)(FQMWHD)(NLPI)(Y) S(2) 62 L (Y)(T)(HR)(S) 103 P P(77)A(12)L(8) 0.17 67 Q (FWH)(Y)(VA)(T) I(2) 10 G (KER)(HD)(Q)(FMW) 167 G E(12)G(70)N(10) 0.17 175 N (Y)(T)(H)(FCWG) A(4).(2) 122 E (FWHR)(KVA)(YCG)(NLPI) 18 T T(89).(2)S(8) 0.18 103 P (YR)(H)(T)(KE) 38 N N(81).(2)S(14) 0.18 167 G (R)(H)(K)(FW) I(2) 18 T (KR)(FMWH)(Q)(LPI) 97 T I(10)T(81)S(6) 0.19 38 N (Y)(H)(FW)(TR) M(2) 97 T (R)(K)(H)(FW) 156 R R(85)D(8)Q(4) 0.19 156 R (TY)(FVCAWDG)(LPI)(SE) S(2) 12 P (R)(Y)(H)(K) 12 P P(87).(4)A(6) 0.20 95 I (R)(Y)(H)(TK) S(2) 102 L (R)(TY)(K)(EH) 95 I I(64)V(33)D(2) 0.20 162 Y (K)(Q)(R)(M) 102 L Y(25)L(64)W(8) 0.21 141 D (R)(H)(FW)(K) M(2) 70 I (YR)(H)(T)(KE) 162 Y A(12)Y(54)C(14) 0.21 155 N (Y)(H)(FTW)(R) L(2)S(14).(2) 141 D D(83)S(4)V(2) 0.22 Table 7. Disruptive mutations for the surface patch in 1ki9A. T(4)G(2)N(2) E(2) 70 I V(10)I(35)L(54) 0.24 155 N N(64)Q(10)S(4) 0.25 3 NOTES ON USING TRACE RESULTS A(14)L(2)H(2) 3.1 Coverage E(2) 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 Table 6. Residues forming surface ”patch” in 1ki9A. 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 Table 7. to some nearby percentage), sorted by the strength of the presumed res type disruptive evolutionary pressure. (I.e., the smaller the coverage, the stronger the mutations pressure on the residue.) Starting from the top of that list, mutating a 43 M (Y)(TH)(SCRG)(FWD) couple of residues should affect the protein somehow, with the exact 92 H (E)(TQMD)(SNKVCLAPIG)(YR) effects to be determined experimentally. 100 G (KER)(FQMWHD)(NYLPI)(SVA) 106 P (YR)(TH)(SKECG)(FQWD) 3.2 Known substitutions continued in next column 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

5 to affect the protein by a point mutation, they should be avoided. For 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- sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with COVERAGE V. The percentage of times the substitution appears in the alignment

is given in the immediately following bracket. No percentage is given V in the cases when it is smaller than 1%. This is meant to be a rough 100% 50% 30% 5% 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 residues that are solvent accessible (according to DSSP program) by V 2 at least 10A˚ , which is roughly the area needed for one water mole- RELATIVE IMPORTANCE cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. Fig. 7. Coloring scheme used to color residues by their relative importance. Note, however, that, if our picture of protein evolution is correct, the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they 4 APPENDIX should not be automatically dropped from consideration when choo- 4.1 File formats sing the set for mutagenesis. (Especially if they form a cluster with Files with extension “ranks sorted” are the actual trace results. The the surface residues.) fields in the table in this file: 3.4 Number of contacts • alignment# number of the position in the alignment Another column worth noting is denoted “noc/bb”; it tells the num- • residue# residue number in the PDB file ber of contacts heavy atoms of the residue in question make across • type amino acid type the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, • rank rank of the position according to older version of ET mutation presumably won’t have strong impact). Two heavy atoms • variability has two subfields: are considered to be “in contact” if their centers are closer than 5A˚ . 1. number of different amino acids appearing in in this column of the alignment 3.5 Annotation 2. their type If the residue annotation is available (either from the pdb file or • 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 • cvg coverage - percentage of the residues on the structure which bond forming residue), and sb (for salt bridge forming residue). have this rho or smaller • gaps percentage of gaps in this column 3.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- 4.2 Color schemes used mentarity with the substitutions found in the alignment. Note that The following color scheme is used in figures with residues colored they are meant to be disruptive to the interaction of the protein by cluster size: black is a single-residue cluster; clusters composed of with its ligand. The attempt is made to complement the following more than one residue colored according to this hierarchy (ordered properties: small [AV GSTC], medium [LPNQDEMIK], large by descending size): red, blue, yellow, green, purple, azure, tur- [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, tively [KHR], or negatively [DE] charged, aromatic [WFYH], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, long aliphatic chain [EKRQM], OH-group possession [SDETY ], DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, and NH2 group possession [NQRK]. The suggestions are listed tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. according to how different they appear to be from the original amino The colors used to distinguish the residues by the estimated acid, and they are grouped in round brackets if they appear equally evolutionary pressure they experience can be seen in Fig. 7. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 4.3 Credits disruptive) These suggestions are tentative - they might prove disrup- 4.3.1 Alistat alistat reads a multiple sequence alignment from the tive to the fold rather than to the interaction. Many researcher will file and shows a number of simple statistics about it. These stati- choose, however, the straightforward alanine mutations, especially in stics include the format, the number of sequences, the total number the beginning stages of their investigation. of residues, the average and range of the sequence lengths, and the

6 alignment length (e.g. including gap characters). Also shown are 4.4 Note about ET Viewer some percent identities. A percent pairwise alignment identity is defi- Dan Morgan from the Lichtarge lab has developed a visualization ned as (idents / MIN(len1, len2)) where idents is the number of tool specifically for viewing trace results. If you are interested, please exact identities and len1, len2 are the unaligned lengths of the two visit: sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, http://mammoth.bcm.tmc.edu/traceview/ and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant The viewer is self-unpacking and self-installing. Input files to be used seq” is calculated by finding the maximum pairwise identity (best with ETV (extension .etvx) can be found in the attachment to the relative) for all N sequences, then finding the minimum of these N main report. numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, 4.5 Citing this work and freely distributed under the GNU General Public License. The method used to rank residues and make predictions in this report can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 4.3.2 CE To map ligand binding sites from different Evolution-Entropy Hybrid Methods for Ranking of Protein Residues source structures, report maker uses the CE program: by Importance” J. Mol. Bio. 336: 1265-82. For the original version http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- ”Protein structure alignment by incremental combinatorial extension tionary Trace Method Defines Binding Surfaces Common to Protein (CE) of the optimal path . Protein Engineering 11(9) 739-747. Families” J. Mol. Bio. 257: 342-358. 4.3.3 DSSP In this work a residue is considered solvent accessi- report maker itself is described in Mihalek I., I. Res and O. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type which is roughly the area needed for one water molecule to come in of service for comparative analysis of proteins.” Bioinformatics the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 22:1656-7. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 4.6 About report maker by [email protected] November 18,2002, report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://www.cmbi.kun.nl/gv/dssp/descrip.html. king visualization program was written by Ivica Res.ˇ report maker 4.3.4 HSSP Whenever available, report maker uses HSSP ali- is copyrighted by Lichtarge Lab, Baylor College of Medicine, gnment as a starting point for the analysis (sequences shorter than Houston. 75% of the query are taken out, however); R. Schneider, A. de 4.7 Attachments Daruvar, and C. Sander. ”The HSSP database of protein structure- The following files should accompany this report: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • http://swift.cmbi.kun.nl/swift/hssp/ 1ki9A.complex.pdb - coordinates of 1ki9A with all of its inter- acting partners A 4.3.5 LaTex The text for this report was processed using LTEX; • 1ki9A.etvx - ET viewer input file for 1ki9A Leslie Lamport, “LaTeX: A Document Preparation System Addison- • Wesley,” Reading, Mass. (1986). 1ki9A.cluster report.summary - Cluster report summary for 1ki9A 4.3.6 Muscle When making alignments “from scratch”, report • 1ki9A.ranks - Ranks file in sequence order for 1ki9A maker uses Muscle alignment program: Edgar, Robert C. (2004), • ”MUSCLE: multiple sequence alignment with high accuracy and 1ki9A.clusters - Cluster descriptions for 1ki9A high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1ki9A.msf - the multiple sequence alignment used for the chain 1ki9A http://www.drive5.com/muscle/ • 1ki9A.descr - description of sequences used in 1ki9A msf 4.3.7 Pymol The figures in this report were produced using • 1ki9A.ranks sorted - full listing of residues and their ranking for Pymol. The scripts can be found in the attachment. Pymol 1ki9A is an open-source application copyrighted by DeLano Scien- • tific LLC (2005). For more information about Pymol see 1ki9A.1ki9C.if.pml - Pymol script for Figure 4 http://pymol.sourceforge.net/. (Note for Windows • 1ki9A.cbcvg - used by other 1ki9A – related pymol scripts users: the attached package needs to be unzipped for Pymol to read • 1ki9A.1ki9B.if.pml - Pymol script for Figure 5 the scripts and launch the viewer.)

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