Pages 1–5 2gkg Evolutionary trace report by report maker April 17, 2009

4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 4.4 Note about ET Viewer 5 4.5 Citing this work 5 4.6 About report maker 5 4.7 Attachments 5

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2gkg): Title: Receiver domain from myxococcus xanthus social motility protein frzs Compound: Mol id: 1; molecule: response regulator homolog; chain: a; fragment: receiver domain (residues 1-124); engineered: CONTENTS yes Organism, scientific name: Myxococcus Xanthus; 1 Introduction 1 2gkg contains a single unique chain 2gkgA (122 residues long). 2 Chain 2gkgA 1 2.1 O68522 overview 1 2.2 Multiple sequence alignment for 2gkgA 1 2.3 Residue ranking in 2gkgA 1 2.4 Top ranking residues in 2gkgA and their position on the structure 1 2 CHAIN 2GKGA 2.4.1 Clustering of residues at 25% coverage. 1 2.1 O68522 overview 2.4.2 Possible novel functional surfaces at 25% From SwissProt, id O68522, 100% identical to 2gkgA: coverage. 2 Description: Response regulator homolog. Organism, scientific name: Myxococcus xanthus. 3 Notes on using trace results 3 Taxonomy: Bacteria; Proteobacteria; Deltaproteobacteria; Myxo- 3.1 Coverage 3 coccales; Cystobacterineae; Myxococcaceae; Myxococcus. 3.2 Known substitutions 3 Subcellular location: Cytoplasmic (By similarity). 3.3 Surface 4 3.4 Number of contacts 4 3.5 Annotation 4 3.6 Mutation suggestions 4 2.2 Multiple sequence alignment for 2gkgA 4 Appendix 4 For the chain 2gkgA, the alignment 2gkgA.msf (attached) with 677 4.1 File formats 4 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 4 database, and fragments shorter than 75% of the query as well as 4.3 Credits 4 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 4 to this report, under the name of 2gkgA.msf. Its statistics, from the 4.3.2 CE 5 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 3-124 in 2gkgA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Format: MSF Number of sequences: 677 Total number of residues: 78066 Smallest: 92 Largest: 122 Average length: 115.3 Alignment length: 122 Average identity: 38% Most related pair: 98% Most unrelated pair: 10% Most distant seq: 33% Fig. 2. Residues in 2gkgA, 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 <1% eukaryotic, and 15% prokaryotic 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 2gkgA.descr. 2.3 Residue ranking in 2gkgA The 2gkgA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2gkgA can be found in the file called 2gkgA.ranks sorted in the attachment. 2.4 Top ranking residues in 2gkgA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 2gkgA 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. 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 Fig. 3. Residues in 2gkgA, colored according to the cluster they belong to: in Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for Table 1. the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. cluster size member color residues red 27 6,8,9,10,11,35,38,47,48,52 53,55,56,62,66,70,79,98,99 Table 1. continued 100,101,102,105,106,107,112 cluster size member continued in next column color residues 115 blue 3 22,26,29

2 Table 1. Clusters of top ranking residues in 2gkgA. Table 2. continued res type substitutions(%) cvg E(2)RTVGF 2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2gkgA surface, away from (or Table 2. Residues forming surface ”patch” in 2gkgA. susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2gkg. It is shown in Fig. 4. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Table 3. res type disruptive mutations 98 H (E)(T)(D)(Q) 102 Y (K)(Q)(E)(MR) 99 A (KE)(R)(Y)(QD) 100 D (R)(H)(FW)(K) 6 L (R)(Y)(H)(T) 70 K (Y)(FW)(T)(VA) 101 E (H)(FWR)(Y)(CG) 47 P (R)(Y)(K)(E) 79 P (Y)(R)(H)(T) 115 R (TD)(Y)(E)(CG) 48 D (R)(FWH)(Y)(K) Fig. 4. A possible active surface on the chain 2gkgA. The larger cluster it belongs to is shown in blue. Table 3. Disruptive mutations for the surface patch in 2gkgA.

The residues belonging to this surface ”patch” are listed in Table Another group of surface residues is shown in Fig.5. The right panel 2, while Table 3 suggests possible disruptive replacements for these shows (in blue) the rest of the larger cluster this surface belongs to. residues (see Section 3.6). Table 2. res type substitutions(%) cvg 98 H G(95)LR(1)QVPHM 0.03 ADE 102 Y F(6)Y(92)CMHV 0.06 99 A A(93)IVC(1)G(2) 0.07 SRTFW 100 D D(91)YKTAV(1) 0.11 S(1)IN(2)RECFHL 6 L L(91)M(2)I(1). 0.12 V(1)FYAHTQ 70 K K(22)R(75)TASQN 0.12 Fig. 5. Another possible active surface on the chain 2gkgA. The larger cluster ECH it belongs to is shown in blue. 101 E E(4)D(83)G(1) 0.16 A(7)QRSFLHYI 47 P P(73)F(5)Y(11) 0.19 The residues belonging to this surface ”patch” are listed in Table A(2)V(2)WI(1) 4, while Table 5 suggests possible disruptive replacements for these H(1)TCDS residues (see Section 3.6). 79 P P(72)V(7)I(12)R 0.20 Table 4. L(2)YA(1)GQM.KF res type substitutions(%) cvg S 105 K K(99)IERL 0.01 115 R R(82)LT(4)A(2) 0.20 106 P P(98)DSAETG 0.02 .(1)HE(1)K(2)MQ 10 S D(97)NP.SGQ 0.05 IVSWC 35 G G(90)VYA(3)F(1) 0.09 48 D D(86)Q(1)S(2) 0.21 SRMILETP H(1)N(2)A(2)K 9 E D(36)E(63).HA 0.13 continued in next column 56 L L(70)M(25)FI(1) 0.14 continued in next column

3 Table 4. continued guide - due to rounding errors these percentages often do not add up res type substitutions(%) cvg to 100%. V(2)T 107 V V(11)F(72)I(6) 0.23 3.3 Surface C(1)Y(4)L(1) To detect candidates for novel functional interfaces, first we look for A(1)ESQTG residues that are solvent accessible (according to DSSP program) by 2 11 D VD(47)E(36)S(8) 0.25 at least 10A˚ , which is roughly the area needed for one water mole- N(3)TIRLQ.YH(1) cule to come in the contact with the residue. Furthermore, we require G that these residues form a “cluster” of residues which have neighbor 55 D .(1)G(7)LM(66) 0.25 within 5A˚ from any of their heavy atoms. S(1)V(5)KE(1) Note, however, that, if our picture of protein evolution is correct, N(7)I(1)THAD(2) the neighboring residues which are not surface accessible might be Q(1)FR equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- Table 4. Residues forming surface ”patch” in 2gkgA. sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 3.4 Number of contacts Table 5. res type disruptive Another column worth noting is denoted “noc/bb”; it tells the num- mutations ber of contacts heavy atoms of the residue in question make across 105 K (Y)(T)(FW)(CG) the interface, as well as how many of them are realized through the 106 P (R)(Y)(H)(K) backbone atoms (if all or most contacts are through the backbone, 10 S (R)(H)(FW)(K) mutation presumably won’t have strong impact). Two heavy atoms 35 G (R)(K)(E)(H) are considered to be “in contact” if their centers are closer than 5A˚ . 9 E (FWH)(R)(Y)(CG) 3.5 Annotation 56 L (R)(Y)(H)(TK) If the residue annotation is available (either from the pdb file or 107 V (R)(K)(E)(Y) from other sources), another column, with the header “annotation” 11 D (R)(H)(FW)(Y) appears. Annotations carried over from PDB are the following: site 55 D (R)(H)(FW)(Y) (indicating existence of related site record in PDB ), S-S (disulfide bond forming residue), hb (hydrogen bond forming residue, jb (james Table 5. Disruptive mutations for the surface patch in 2gkgA. bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 3.1 Coverage they are meant to be disruptive to the interaction of the protein Trace results are commonly expressed in terms of coverage: the resi- with its ligand. The attempt is made to complement the following due is important if its “coverage” is small - that is if it belongs to properties: small [AV GSTC], medium [LPNQDEMIK], large some small top percentage of residues [100% is all of the residues [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [WFYH], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDETY ], to some nearby percentage), sorted by the strength of the presumed and NH2 group possession [NQRK]. The suggestions are listed evolutionary pressure. (I.e., the smaller the coverage, the stronger the according to how different they appear to be from the original amino pressure on the residue.) Starting from the top of that list, mutating a acid, and they are grouped in round brackets if they appear equally couple of residues should affect the protein somehow, with the exact disruptive. From left to right, each bracketed group of amino acid effects to be determined experimentally. types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will One of the table columns is “substitutions” - other amino acid types choose, however, the straightforward alanine mutations, especially in seen at the same position in the alignment. These amino acid types the beginning stages of their investigation. 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 4 APPENDIX 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- 4.1 File formats sely, when looking for substitutions which will not affect the protein, Files with extension “ranks sorted” are the actual trace results. The one may try replacing, R with K, or (perhaps more surprisingly), with fields in the table in this file: V. The percentage of times the substitution appears in the alignment • is given in the immediately following bracket. No percentage is given alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough • residue# residue number in the PDB file

4 seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, COVERAGE and freely distributed under the GNU General Public License. 4.3.2 CE To map ligand binding sites from different V source structures, report maker uses the CE program: 100% 50% 30% 5% http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.3.3 DSSP In this work a residue is considered solvent accessi- ble if the DSSP program finds it exposed to water by at least 10A˚ 2, V which is roughly the area needed for one water molecule to come in RELATIVE IMPORTANCE the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, Fig. 6. Coloring scheme used to color residues by their relative importance. http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • type amino acid type gnment as a starting point for the analysis (sequences shorter than • rank rank of the position according to older version of ET 75% of the query are taken out, however); R. Schneider, A. de • variability has two subfields: Daruvar, and C. Sander. ”The HSSP database of protein structure- 1. number of different amino acids appearing in in this column sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. of the alignment http://swift.cmbi.kun.nl/swift/hssp/ 2. their type 4.3.5 LaTex The text for this report was processed using LAT X; • E rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 6. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version

5 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 2gkgA.complex.pdb - coordinates of 2gkgA with all of its tionary Trace Method Defines Binding Surfaces Common to Protein interacting partners Families” J. Mol. Bio. 257: 342-358. • 2gkgA.etvx - ET viewer input file for 2gkgA report maker itself is described in Mihalek I., I. Res and O. • 2gkgA.cluster report.summary - Cluster report summary for Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 2gkgA of service for comparative analysis of proteins.” Bioinformatics 22:1656-7. • 2gkgA.ranks - Ranks file in sequence order for 2gkgA • 2gkgA.clusters - Cluster descriptions for 2gkgA 4.6 About report maker • 2gkgA.msf - the multiple sequence alignment used for the chain report maker was written in 2006 by Ivana Mihalek. The 1D ran- 2gkgA king visualization program was written by Ivica Res.ˇ report maker • is copyrighted by Lichtarge Lab, Baylor College of Medicine, 2gkgA.descr - description of sequences used in 2gkgA msf Houston. • 2gkgA.ranks sorted - full listing of residues and their ranking for 2gkgA 4.7 Attachments The following files should accompany this report:

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