Pages 1–6 1a68 Evolutionary trace report by report maker June 1, 2010

4.3.1 Alistat 5 4.3.2 CE 5 4.3.3 DSSP 5 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 6 4.7 Attachments 6

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1a68): Title: Crystal structure of the tetramerization domain of the shaker potassium channel CONTENTS Compound: Mol id: 1; molecule: potassium channel kv1.1; chain: a; fragment: tetramerization domain; engineered: yes; mutation: yes 1 Introduction 1 Organism, scientific name: Californica; 1a68 contains a single unique chain 1a68A (87 residues long). 2 Chain 1a68A 1 2.1 Q16968 overview 1 2.2 Multiple sequence alignment for 1a68A 1 2.3 Residue ranking in 1a68A 1 2.4 Top ranking residues in 1a68A and their position on 2 CHAIN 1A68A the structure 1 2.1 Q16968 overview 2.4.1 Clustering of residues at 25% coverage. 1 2.4.2 Overlap with known functional surfaces at From SwissProt, id Q16968, 100% identical to 1a68A: 25% coverage. 2 Description: Potassium channel. 2.4.3 Possible novel functional surfaces at 25% Organism, scientific name: Aplysia californica (California sea coverage. 3 hare). Taxonomy: Eukaryota; Metazoa; ; ; Ortho- 3 Notes on using trace results 4 gastropoda; ; ; Euthyneura; Opistho- 3.1 Coverage 4 branchia; ; ; ; Aplysia. 3.2 Known substitutions 4 3.3 Surface 4 3.4 Number of contacts 5 3.5 Annotation 5 2.2 Multiple sequence alignment for 1a68A 3.6 Mutation suggestions 5 For the chain 1a68A, the alignment 1a68A.msf (attached) with 251 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 5 to this report, under the name of 1a68A.msf. Its statistics, from the 4.3 Credits 5 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 66-152 in 1a68A colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Format: MSF Number of sequences: 251 Total number of residues: 21289 Smallest: 67 Largest: 87 Average length: 84.8 Alignment length: 87 Average identity: 43% Most related pair: 99% Most unrelated pair: 15% Most distant seq: 35%

Furthermore, <1% of residues show as conserved in this ali- Fig. 2. Residues in 1a68A, colored by their relative importance. Clockwise: gnment. front, back, top and bottom views. The alignment consists of 51% eukaryotic ( 36% vertebrata, 3% arthropoda, <1% 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 1a68A.descr.

2.3 Residue ranking in 1a68A The 1a68A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1a68A can be found in the file called 1a68A.ranks sorted in the attachment.

2.4 Top ranking residues in 1a68A 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 1a68A 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 1a68A, colored according to the cluster they belong to: in Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The Table 1. corresponding Pymol script is attached. cluster size member color residues red 19 71,73,74,83,84,91,92,108,110 2.4.2 Overlap with known functional surfaces at 25% coverage. 111,112,113,115,118,122,125 The name of the ligand is composed of the source PDB identifier 145,148,149 and the heteroatom name used in that file. blue 2 129,131 Interface with 1a68A2.Table 2 lists the top 25% of residues at the interface with 1a68A2. The following table (Table 3) suggests Table 1. Clusters of top ranking residues in 1a68A. possible disruptive replacements for these residues (see Section 3.6).

2 Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 113 R R(98)VL 0.01 14/14 3.61 HG 112 D D(94) 0.03 64/12 2.72 N(1) E(3)V 74 G G(98) 0.05 28/28 3.84 .(1)V 110 F F(89) 0.06 52/0 3.57 Y(8)TRD VC 145 E E(96) 0.07 2/0 4.79 D(3). 71 N N(96) 0.08 10/0 3.38 .(1)KIS 108 E E(94)Q. 0.15 2/0 4.34 MVAR(2) ND 115 R P(56) 0.18 50/1 3.15 S(3) R(33) K(1)E G(3) 73 S G(48) 0.25 39/29 2.75 S(44) A(3) .(1) R(1)Y

Table 2. The top 25% of residues in 1a68A at the interface with 1a68A2. (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 113 R (D)(E)(T)(Y) 112 D (R)(H)(FW)(Y) 74 G (KER)(HD)(Q)(FMW) 110 F (K)(E)(Q)(D) 145 E (FW)(H)(VCAG)(R) 71 N (Y)(FWH)(T)(CRG) 108 E (H)(FYW)(CRG)(TVA) 115 R (Y)(T)(D)(FW) 73 S (K)(R)(M)(Q)

Table 3. List of disruptive mutations for the top 25% of residues in 1a68A, that are at the interface with 1a68A2.

Figure 4 shows residues in 1a68A colored by their importance, at the interface with 1a68A2.

3 Fig. 4. Residues in 1a68A, at the interface with 1a68A2, colored by their rela- Fig. 5. Residues in 1a68A, at the interface with 1a68A3, colored by their rela- tive importance. 1a68A2 is shown in backbone representation (See Appendix tive importance. 1a68A3 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1a68A.) for the coloring scheme for the protein chain 1a68A.)

Figure 5 shows residues in 1a68A colored by their importance, at the Interface with 1a68A3.Table 4 lists the top 25% of residues at interface with 1a68A3. the interface with 1a68A3. The following table (Table 5) suggests possible disruptive replacements for these residues (see Section 3.6). 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1a68A surface, away from (or Table 4. susbtantially larger than) other functional sites and interfaces reco- res type subst’s cvg noc/ dist gnizable in PDB entry 1a68. It is shown in Fig. 6. The right panel (%) bb (A˚ ) shows (in blue) the rest of the larger cluster this surface belongs to. 83 T T(88) 0.14 9/0 2.72 A(4) L(1)NEQ FSVI 122 L L(92) 0.16 4/0 4.03 F(1). Y(1) M(1)VWI

Table 4. The top 25% of residues in 1a68A at the interface with 1a68A3. (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. 6. A possible active surface on the chain 1a68A. The larger cluster it belongs to is shown in blue.

Table 5. The residues belonging to this surface ”patch” are listed in Table res type disruptive 6, while Table 7 suggests possible disruptive replacements for these mutations residues (see Section 3.6). 83 T (R)(K)(H)(FW) 122 L (R)(Y)(T)(K)

Table 5. List of disruptive mutations for the top 25% of residues in 1a68A, that are at the interface with 1a68A3.

4 Table 6. evolutionary pressure. (I.e., the smaller the coverage, the stronger the res type substitutions(%) cvg pressure on the residue.) Starting from the top of that list, mutating a 113 R R(98)VLHG 0.01 couple of residues should affect the protein somehow, with the exact 118 F F(99).A 0.02 effects to be determined experimentally. 112 D D(94)N(1)E(3)V 0.03 74 G G(98).(1)V 0.05 3.2 Known substitutions 110 F F(89)Y(8)TRDVC 0.06 One of the table columns is “substitutions” - other amino acid types 145 E E(96)D(3). 0.07 seen at the same position in the alignment. These amino acid types 71 N N(96).(1)KIS 0.08 may be interchangeable at that position in the protein, so if one wants 111 F F(92)VL(1)AI(5) 0.10 to affect the protein by a point mutation, they should be avoided. For 108 E E(94)Q.MVAR(2)N 0.15 example if the substitutions are “RVK” and the original protein has D an R at that position, it is advisable to try anything, but RVK. Conver- 122 L L(92)F(1).Y(1) 0.16 sely, when looking for substitutions which will not affect the protein, M(1)VWI one may try replacing, R with K, or (perhaps more surprisingly), with 115 R P(56)S(3)R(33) 0.18 V. The percentage of times the substitution appears in the alignment K(1)EG(3) is given in the immediately following bracket. No percentage is given 91 L R(41)L(51)SP 0.20 in the cases when it is smaller than 1%. This is meant to be a rough M(3)V(1) guide - due to rounding errors these percentages often do not add up 148 F Y(31)F(67).(1) 0.23 to 100%. 149 Y W(43)F(18)Y(36) 0.24 .(1) 3.3 Surface 73 S G(48)S(44)A(3) 0.25 To detect candidates for novel functional interfaces, first we look for .(1)R(1)Y residues that are solvent accessible (according to DSSP program) by 2 at least 10A˚ , which is roughly the area needed for one water mole- Table 6. Residues forming surface ”patch” in 1a68A. 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. Table 7. Note, however, that, if our picture of protein evolution is correct, res type disruptive the neighboring residues which are not surface accessible might be mutations equally important in maintaining the interaction specificity - they 113 R (D)(E)(T)(Y) should not be automatically dropped from consideration when choo- 118 F (E)(K)(D)(Q) sing the set for mutagenesis. (Especially if they form a cluster with 112 D (R)(H)(FW)(Y) the surface residues.) 74 G (KER)(HD)(Q)(FMW) 3.4 Number of contacts 110 F (K)(E)(Q)(D) 145 E (FW)(H)(VCAG)(R) Another column worth noting is denoted “noc/bb”; it tells the num- 71 N (Y)(FWH)(T)(CRG) ber of contacts heavy atoms of the residue in question make across 111 F (KE)(R)(Q)(TD) the interface, as well as how many of them are realized through the 108 E (H)(FYW)(CRG)(TVA) backbone atoms (if all or most contacts are through the backbone, 122 L (R)(Y)(T)(K) mutation presumably won’t have strong impact). Two heavy atoms ˚ 115 R (Y)(T)(D)(FW) are considered to be “in contact” if their centers are closer than 5A. 91 L (Y)(R)(H)(T) 3.5 Annotation 148 F (K)(E)(Q)(D) 149 Y (K)(Q)(EM)(N) If the residue annotation is available (either from the pdb file or 73 S (K)(R)(M)(Q) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 7. Disruptive mutations for the surface patch in 1a68A. bond forming residue), hb (hydrogen bond forming residue, jb (james 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- 3.1 Coverage mentarity with the substitutions found in the alignment. Note that Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ],

5 bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated COVERAGE evolutionary pressure they experience can be seen in Fig. 7. 4.3 Credits V 4.3.1 Alistat alistat reads a multiple sequence alignment from the 100% 50% 30% 5% file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi-

V ned as (idents / MIN(len1, len2)) where idents is the number of RELATIVE IMPORTANCE exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, Fig. 7. Coloring scheme used to color residues by their relative importance. and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N and NH2 group possession [NQRK]. The suggestions are listed numbers (hence, the most outlying sequence). alistat is copyrighted according to how different they appear to be from the original amino by HHMI/Washington University School of Medicine, 1992-2001, acid, and they are grouped in round brackets if they appear equally and freely distributed under the GNU General Public License. disruptive. From left to right, each bracketed group of amino acid 4.3.2 CE To map ligand binding sites from different types resembles more strongly the original (i.e. is, presumably, less source structures, report maker uses the CE program: disruptive) These suggestions are tentative - they might prove disrup- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tive to the fold rather than to the interaction. Many researcher will ”Protein structure alignment by incremental combinatorial extension choose, however, the straightforward alanine mutations, especially in (CE) of the optimal path . Protein Engineering 11(9) 739-747. the beginning stages of their investigation. 4.3.3 DSSP In this work a residue is considered solvent accessi- ˚ 2 4 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A , which is roughly the area needed for one water molecule to come in 4.1 File formats the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Files with extension “ranks sorted” are the actual trace results. The Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version fields in the table in this file: by [email protected] November 18,2002,

• alignment# number of the position in the alignment http://www.cmbi.kun.nl/gv/dssp/descrip.html. • residue# residue number in the PDB file 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 75% of the query are taken out, however); R. Schneider, A. de • rank rank of the position according to older version of ET Daruvar, and C. Sander. ”The HSSP database of protein structure- • variability has two subfields: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 1. number of different amino acids appearing in in this column 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 LATEX; • 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

6 users: the attached package needs to be unzipped for Pymol to read 4.6 About report maker the scripts and launch the viewer.) report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.4 Note about ET Viewer is copyrighted by Lichtarge Lab, Baylor College of Medicine, Dan Morgan from the Lichtarge lab has developed a visualization Houston. tool specifically for viewing trace results. If you are interested, please 4.7 Attachments visit: The following files should accompany this report: • http://mammoth.bcm.tmc.edu/traceview/ 1a68A.complex.pdb - coordinates of 1a68A with all of its interacting partners • 1a68A.etvx - ET viewer input file for 1a68A The viewer is self-unpacking and self-installing. Input files to be used with ETV (extension .etvx) can be found in the attachment to the • 1a68A.cluster report.summary - Cluster report summary for main report. 1a68A • 1a68A.ranks - Ranks file in sequence order for 1a68A 4.5 Citing this work • 1a68A.clusters - Cluster descriptions for 1a68A The method used to rank residues and make predictions in this report • 1a68A.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 1a68A Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1a68A.descr - description of sequences used in 1a68A msf by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1a68A.ranks sorted - full listing of residues and their ranking of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- for 1a68A tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. • 1a68A.1a68A2.if.pml - Pymol script for Figure 4 report maker itself is described in Mihalek I., I. Res and O. • 1a68A.cbcvg - used by other 1a68A – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1a68A.1a68A3.if.pml - Pymol script for Figure 5 of service for comparative analysis of proteins.” Bioinformatics 22:1656-7.

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