Pages 1–7 2b05 Evolutionary trace report by report maker July 3, 2010

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

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2b05): Title: Crystal structure of 14-3-3 gamma in complex with a phospho- serine peptide Compound: Mol id: 1; molecule: 14-3-3 protein gamma; chain: a, b, c, d, e, f; synonym: protein kinase c inhibitor protein 1, kcip-1; engineered: yes; mol id: 2; molecule: peptide; chain: g, h, i, j, k, l; CONTENTS engineered: yes Organism, scientific name: Homo Sapiens; 1 Introduction 1 2b05 contains a single unique chain 2b05A (233 residues long) and its homologues 2b05F, 2b05E, 2b05B, 2b05C, and 2b05D. Chains 2 Chain 2b05A 1 2b05G, 2b05H, 2b05I, 2b05J, 2b05K, and 2b05L are too short to 2.1 Q6FH52 overview 1 permit statistically significant analysis, and were treated as a peptide 2.2 Multiple sequence alignment for 2b05A 1 ligands. 2.3 Residue ranking in 2b05A 1 2.4 Top ranking residues in 2b05A and their position on the structure 1 2 CHAIN 2B05A 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Overlap with known functional surfaces at 2.1 Q6FH52 overview 25% coverage. 2 From SwissProt, id Q6FH52, 100% identical to 2b05A: 2.4.3 Possible novel functional surfaces at 25% Description: YWHAG protein. coverage. 4 Organism, scientific name: Homo sapiens (Human). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3 Notes on using trace results 5 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 3.1 Coverage 5 Catarrhini; Hominidae; Homo. 3.2 Known substitutions 5 Similarity: Belongs to the 14-3-3 family. 3.3 Surface 6 3.4 Number of contacts 6 3.5 Annotation 6 2.2 Multiple sequence alignment for 2b05A 3.6 Mutation suggestions 6 For the chain 2b05A, the alignment 2b05A.msf (attached) with 470 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 6 database, and fragments shorter than 75% of the query as well as 4.1 File formats 6 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 6 to this report, under the name of 2b05A.msf. Its statistics, from the 4.3 Credits 6 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 2-117 in 2b05A colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Fig. 2. Residues 118-234 in 2b05A colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.) Fig. 3. Residues in 2b05A, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 470 Total number of residues: 100908 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 62 top 25% of all residues, this time colored according to clusters they Largest: 233 belong to. The clusters in Fig.4 are composed of the residues listed Average length: 214.7 Alignment length: 233 Average identity: 61% Most related pair: 99% Most unrelated pair: 0% Most distant seq: 38%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 49% eukaryotic ( 10% vertebrata, 1% arthropoda, 5% fungi, 20% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 2b05A.descr.

2.3 Residue ranking in 2b05A The 2b05A sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 2b05A can be found in the file called 2b05A.ranks sorted in the attachment.

2.4 Top ranking residues in 2b05A and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 2b05A, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for residues in the protein . Figure 3 shows residues in 2b05A colored the coloring scheme). Clockwise: front, back, top and bottom views. The by their importance: bright red and yellow indicate more conser- corresponding Pymol script is attached. ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1.

2 Table 1. cluster size member color residues red 56 23,37,41,42,43,44,45,46,47 48,49,50,56,57,59,60,61,90 92,93,97,100,101,108,121,122 123,125,126,127,128,129,130 132,133,135,136,154,157,161 170,172,173,174,175,177,178 180,181,182,185,186,193,194 197,222

Table 1. Clusters of top ranking residues in 2b05A.

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 2b05D.Table 2 lists the top 25% of residues at the interface with 2b05D. 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˚ ) 92 E E(94)KA 0.05 12/0 2.80 .(4)DYS GQ 77 K 50.(5)7 0.06 5/4 3.35 4 59 S S(72) 0.16 21/8 2.94 G(1) A(17) .(4)C E(1)VPT WQ 85 Y Y(86) 0.16 62/0 2.55 .(5)V F(4)TCQ MIHL(1) PK 56 R R(81)HG 0.20 5/0 4.23 L(5) .(5)A K(3)DTP Q(1)YN

Table 2. The top 25% of residues in 2b05A at the interface with 2b05D. (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. )

3 Table 3. Table 4. continued res type disruptive res type subst’s cvg noc/ dist mutations (%) bb (A˚ ) 92 E (FWH)(R)(Y)(VA) GSK 77 K (Y)(T)(FW)(S) 50 K K(93) 0.02 22/0 2.78 59 S (R)(K)(H)(FQW) .(5)CAG 85 Y (K)(QR)(E)(M) N 56 R (T)(D)(Y)(E) 129 D D(95)Q 0.02 2/0 4.86 .(2)PTN Table 3. List of disruptive mutations for the top 25% of residues in VM 2b05A, that are at the interface with 2b05D. 57 R R(94) 0.03 14/0 2.65 .(5)LAP 125 K K(94) 0.05 6/0 3.92 .(3)RPG ENST 174 G G(93)Q 0.07 6/6 4.34 .(4)VPS TAY 181 V V(92)I 0.12 13/0 3.68 .(5)LEA SF 178 N N(92)D 0.14 25/0 2.76 .(5)ELH RSKQ 177 L L(93)F 0.15 16/0 3.48 .(5)GSP N 222 I I(89) 0.22 4/0 3.84 .(8)TVG MS 185 E E(91)VG 0.24 1/0 4.71 K.(6)DQ XF

Table 4. The top 25% of residues in 2b05A at the interface with 2b05G. (Field names: res: residue number in the PDB entry; type: amino acid type; Fig. 5. Residues in 2b05A, at the interface with 2b05D, colored by their rela- substs: substitutions seen in the alignment; with the percentage of each type tive importance. 2b05D is shown in backbone representation (See Appendix in the bracket; noc/bb: number of contacts with the ligand, with the number of for the coloring scheme for the protein chain 2b05A.) contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Figure 5 shows residues in 2b05A colored by their importance, at the interface with 2b05D. Table 5. Interface with the peptide 2b05G. Table 4 lists the top 25% res type disruptive of residues at the interface with 2b05G. The following table (Table mutations 5) suggests possible disruptive replacements for these residues (see 132 R (TYD)(FVLAWPI)(ECG)(S) Section 3.6). 133 Y (K)(QM)(E)(R) Table 4. 136 E (H)(FW)(Y)(R) res type subst’s cvg noc/ dist 50 K (Y)(FW)(T)(H) (%) bb (A˚ ) 129 D (R)(H)(FW)(Y) 132 R R(95)Q 0.00 16/0 2.60 57 R (T)(YD)(E)(S) .(3)DSC 125 K (Y)(FW)(T)(VAH) 133 Y Y(94)V 0.01 16/0 2.45 174 G (R)(K)(E)(H) .(3)INR 181 V (R)(KY)(E)(H) 136 E E(94)L 0.01 1/0 4.44 178 N (Y)(FTW)(H)(CG) .(3)PNQ 177 L (R)(Y)(H)(T) continued in next column continued in next column

4 Table 5. continued res type disruptive mutations 222 I (R)(Y)(H)(K) 185 E (H)(FW)(Y)(R)

Table 5. List of disruptive mutations for the top 25% of residues in 2b05A, that are at the interface with 2b05G.

Fig. 7. A possible active surface on the chain 2b05A. The larger cluster it belongs to is shown in blue.

Table 6. continued res type substitutions(%) cvg 57 R R(94).(5)LAP 0.03 130 Y Y(95)V.(3)IFCHA 0.03 Q 49 Y Y(90).(5)F(2)KT 0.04 LIR 92 E E(94)KA.(4)DYSG 0.05 Q 125 K K(94).(3)RPGENS 0.05 T 126 M M(91)L(3).(2)T 0.06 I(1)VNK 122 F F(92).(3)Y(1) 0.07 L(1)SI 174 G G(93)Q.(4)VPSTA 0.07 Y 37 L L(90).(8)MGFS 0.08 Fig. 6. Residues in 2b05A, at the interface with 2b05G, colored by their rela- 61 R R(92).(5)NGKLVA 0.08 tive importance. 2b05G is shown in backbone representation (See Appendix HSTE for the coloring scheme for the protein chain 2b05A.) 46 S S(91).(7)VMTA 0.09 41 E E(87).(7)QD(2)H 0.10 Figure 6 shows residues in 2b05A colored by their importance, at the ARS interface with 2b05G. 44 L L(89).(7)STFA 0.11 I(1)M 2.4.3 Possible novel functional surfaces at 25% coverage. One 42 R R(90).(7)TEAKI 0.12 group of residues is conserved on the 2b05A surface, away from (or 181 V V(92)I.(5)LEASF 0.12 susbtantially larger than) other functional sites and interfaces reco- 108 L L(91)C(1)I(1)VY 0.13 gnizable in PDB entry 2b05. It is shown in Fig. 7. The right panel .(3)FMAQ shows (in blue) the rest of the larger cluster this surface belongs to. 43 N N(87).(7)LAS(1) 0.14 The residues belonging to this surface ”patch” are listed in Table DHTQKIPR 6, while Table 7 suggests possible disruptive replacements for these 178 N N(92)D.(5)ELHRS 0.14 residues (see Section 3.6). KQ Table 6. 47 V V(87).(5)I(2)SY 0.15 res type substitutions(%) cvg A(1)XCTHMNF 132 R R(95)Q.(3)DSC 0.00 60 W W(87)GVI(1).(4) 0.15 133 Y Y(94)V.(3)INR 0.01 RL(4)MHCYN 136 E E(94)L.(3)PNQGS 0.01 177 L L(93)F.(5)GSPN 0.15 K 59 S S(72)G(1)A(17) 0.16 50 K K(93).(5)CAGN 0.02 continued in next column 129 D D(95)Q.(2)PTNVM 0.02 continued in next column

5 Table 6. continued Table 7. continued res type substitutions(%) cvg res type disruptive .(4)CE(1)VPTWQ mutations 101 L L(83)M(5)I(4) 0.17 43 N (Y)(H)(FW)(T) V(1).(3)NCFTP 178 N (Y)(FTW)(H)(CG) 186 I I(87)V(5)E.(6)T 0.18 47 V (KR)(E)(Y)(Q) MNL 60 W (E)(K)(D)(T) 127 K K(87)R(1)QYT 0.19 177 L (R)(Y)(H)(T) M(2).(3)E(1)HLN 59 S (R)(K)(H)(FQW) VAP 101 L (R)(Y)(H)(T) 56 R R(81)HGL(5).(5) 0.20 186 I (YR)(H)(T)(K) AK(3)DTPQ(1)YN 127 K (Y)(T)(FW)(CG) 100 V I(61)V(30).(3)F 0.21 56 R (T)(D)(Y)(E) L(2)TGYS 100 V (KR)(E)(Q)(Y) 222 I I(89).(8)TVGMS 0.22 222 I (R)(Y)(H)(K) 90 E E(89).(4)V(1)SA 0.23 90 E (H)(FW)(Y)(R) QFKICNTRGL 172 R (D)(T)(YE)(CG) 172 R R(89)A(1)I.(4) 0.23 123 Y (K)(Q)(E)(M) Y(1)MKTS(1)NHWQ 170 P (Y)(R)(H)(T) 123 Y Y(86)L(1)F(6)C 0.24 185 E (H)(FW)(Y)(R) .(3)SHIWR 169 H (E)(T)(MD)(Q) 170 P P(91)L.(4)SAMVT 0.24 NQH Table 7. Disruptive mutations for the surface patch in 2b05A. 185 E E(91)VGK.(6)DQX 0.24 F 169 H H(81)N(6).(4) 0.25 S(2)D(2)RTKLGQ 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Table 6. Residues forming surface ”patch” in 2b05A. 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 Table 7. in a chain], according to trace. The ET results are presented in the res type disruptive form of a table, usually limited to top 25% percent of residues (or mutations to some nearby percentage), sorted by the strength of the presumed 132 R (TYD)(FVLAWPI)(ECG)(S) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 133 Y (K)(QM)(E)(R) pressure on the residue.) Starting from the top of that list, mutating a 136 E (H)(FW)(Y)(R) couple of residues should affect the protein somehow, with the exact 50 K (Y)(FW)(T)(H) effects to be determined experimentally. 129 D (R)(H)(FW)(Y) 57 R (T)(YD)(E)(S) 3.2 Known substitutions 130 Y (K)(Q)(E)(R) One of the table columns is “substitutions” - other amino acid types 49 Y (K)(Q)(EM)(R) seen at the same position in the alignment. These amino acid types 92 E (FWH)(R)(Y)(VA) may be interchangeable at that position in the protein, so if one wants 125 K (Y)(FW)(T)(VAH) to affect the protein by a point mutation, they should be avoided. For 126 M (Y)(H)(T)(R) example if the substitutions are “RVK” and the original protein has 122 F (K)(E)(Q)(R) an R at that position, it is advisable to try anything, but RVK. Conver- 174 G (R)(K)(E)(H) sely, when looking for substitutions which will not affect the protein, 37 L (R)(Y)(H)(TK) one may try replacing, R with K, or (perhaps more surprisingly), with 61 R (D)(Y)(T)(E) V. The percentage of times the substitution appears in the alignment 46 S (R)(K)(H)(Q) is given in the immediately following bracket. No percentage is given 41 E (FW)(H)(Y)(CG) in the cases when it is smaller than 1%. This is meant to be a rough 44 L (R)(Y)(H)(K) guide - due to rounding errors these percentages often do not add up 42 R (TY)(D)(CG)(E) to 100%. 181 V (R)(KY)(E)(H) 108 L (R)(Y)(H)(T) 3.3 Surface continued in next column To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require

6 that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. 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 COVERAGE should not be automatically dropped from consideration when choo-

sing the set for mutagenesis. (Especially if they form a cluster with V the surface residues.) 100% 50% 30% 5% 3.4 Number of contacts Another column worth noting is denoted “noc/bb”; it tells the num- 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 backbone atoms (if all or most contacts are through the backbone, V mutation presumably won’t have strong impact). Two heavy atoms RELATIVE IMPORTANCE are considered to be “in contact” if their centers are closer than 5A˚ .

3.5 Annotation Fig. 8. Coloring scheme used to color residues by their relative importance. If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller • gaps Mutation suggestions are completely heuristic and based on comple- percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [WFYH], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDETY ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 8. disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will 4.3.1 Alistat alistat reads a multiple sequence alignment from the choose, however, the straightforward alanine mutations, especially in file and shows a number of simple statistics about it. These stati- the beginning stages of their investigation. stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 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:

7 http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) The viewer is self-unpacking and self-installing. Input files to be used ”Protein structure alignment by incremental combinatorial extension with ETV (extension .etvx) can be found in the attachment to the (CE) of the optimal path . Protein Engineering 11(9) 739-747. main report. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4.5 Citing this work 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , The method used to rank residues and make predictions in this report which is roughly the area needed for one water molecule to come in can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by Importance” J. Mol. Bio. 336: 1265-82. For the original version by [email protected] November 18,2002, of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- http://www.cmbi.kun.nl/gv/dssp/descrip.html. tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- report maker itself is described in Mihalek I., I. Res and O. gnment as a starting point for the analysis (sequences shorter than Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 75% of the query are taken out, however); R. Schneider, A. de of service for comparative analysis of proteins.” Bioinformatics Daruvar, and C. Sander. ”The HSSP database of protein structure- 22:1656-7. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 4.6 About report maker report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://swift.cmbi.kun.nl/swift/hssp/ king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- Houston. Wesley,” Reading, Mass. (1986). 4.7 Attachments 4.3.6 Muscle When making alignments “from scratch”, report The following files should accompany this report: maker uses Muscle alignment program: Edgar, Robert C. (2004), • 2b05A.complex.pdb - coordinates of 2b05A with all of its ”MUSCLE: multiple sequence alignment with high accuracy and interacting partners high throughput.” Nucleic Acids Research 32(5), 1792-97. • 2b05A.etvx - ET viewer input file for 2b05A http://www.drive5.com/muscle/ • 2b05A.cluster report.summary - Cluster report summary for 4.3.7 Pymol The figures in this report were produced using 2b05A Pymol. The scripts can be found in the attachment. Pymol • 2b05A.ranks - Ranks file in sequence order for 2b05A is an open-source application copyrighted by DeLano Scien- • 2b05A.clusters - Cluster descriptions for 2b05A tific LLC (2005). For more information about Pymol see • 2b05A.msf - the multiple sequence alignment used for the chain http://pymol.sourceforge.net/. (Note for Windows 2b05A users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) • 2b05A.descr - description of sequences used in 2b05A msf • 2b05A.ranks sorted - full listing of residues and their ranking 4.4 Note about ET Viewer for 2b05A Dan Morgan from the Lichtarge lab has developed a visualization • 2b05A.2b05D.if.pml - Pymol script for Figure 5 tool specifically for viewing trace results. If you are interested, please • visit: 2b05A.cbcvg - used by other 2b05A – related pymol scripts • 2b05A.2b05G.if.pml - Pymol script for Figure 6 http://mammoth.bcm.tmc.edu/traceview/

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