Pages 1–5 1iu9 Evolutionary trace report by report maker July 1, 2010

4.3.3 DSSP 4 4.3.4 HSSP 4 4.3.5 LaTex 4 4.3.6 Muscle 4 4.3.7 Pymol 4 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 1iu9): Title: Crystal structure of the c-terminal domain of aspartate race- mase from horikoshii ot3 Compound: Mol id: 1; molecule: aspartate racemase; chain: a; fragment: c-terminal domain; ec: 5.1.1.13; engineered: yes Organism, scientific name: ; 1iu9 contains a single unique chain 1iu9A (111 residues long).

2 CHAIN 1IU9A 2.1 O58403 overview CONTENTS From SwissProt, id O58403, 100% identical to 1iu9A: 1 Introduction 1 Description: 228aa long hypothetical aspartate racemase. Organism, scientific name: Pyrococcus horikoshii. 2 Chain 1iu9A 1 : ; ; ; Thermococca- 2.1 O58403 overview 1 les; Thermococcaceae; Pyrococcus. 2.2 Multiple sequence alignment for 1iu9A 1 2.3 Residue ranking in 1iu9A 1 2.2 Multiple sequence alignment for 1iu9A 2.4 Top ranking residues in 1iu9A and their position on For the chain 1iu9A, the alignment 1iu9A.msf (attached) with 187 the structure 1 sequences was used. The alignment was downloaded from the HSSP 2.4.1 Clustering of residues at 25% coverage. 1 database, and fragments shorter than 75% of the query as well as 2.4.2 Possible novel functional surfaces at 25% duplicate sequences were removed. It can be found in the attachment coverage. 2 to this report, under the name of 1iu9A.msf. Its statistics, from the alistat program are the following: 3 Notes on using trace results 3 3.1 Coverage 3 Format: MSF 3.2 Known substitutions 3 Number of sequences: 187 3.3 Surface 3 Total number of residues: 20223 3.4 Number of contacts 3 Smallest: 86 3.5 Annotation 3 Largest: 111 3.6 Mutation suggestions 3 Average length: 108.1 Alignment length: 111 4 Appendix 4 Average identity: 32% 4.1 File formats 4 Most related pair: 99% 4.2 Color schemes used 4 Most unrelated pair: 7% 4.3 Credits 4 Most distant seq: 31% 4.3.1 Alistat 4 4.3.2 CE 4

1 Lichtarge lab 2006 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. 1. Residues 103-213 in 1iu9A colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.)

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 17% prokaryotic, and 6% 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 1iu9A.descr. 2.3 Residue ranking in 1iu9A The 1iu9A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1iu9A can be found in the file called 1iu9A.ranks sorted in the attachment. 2.4 Top ranking residues in 1iu9A and their position on the structure In the following we consider residues ranking among top 25% of resi- Fig. 3. Residues in 1iu9A, colored according to the cluster they belong to: dues in the protein . Figure 2 shows residues in 1iu9A colored by their red, followed by blue and yellow are the largest clusters (see Appendix for importance: bright red and yellow indicate more conserved/important the coloring scheme). Clockwise: front, back, top and bottom views. The residues (see Appendix for the coloring scheme). A Pymol script for corresponding Pymol script is attached. producing this figure can be found in the attachment. in Table 1. Table 1. cluster size member color residues red 26 120,121,122,123,124,126,127 128,132,133,147,159,160,163 164,166,191,192,193,194,195 196,197,198,200,212

Table 1. Clusters of top ranking residues in 1iu9A.

2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1iu9A surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1iu9. It is shown in Fig. 4. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 2, while Table 3 suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type substitutions(%) cvg Fig. 2. Residues in 1iu9A, colored by their relative importance. Clockwise: 194 C C(98).S 0.01 front, back, top and bottom views. continued in next column

2 Table 3. res type disruptive mutations 194 C (KR)(E)(FMWH)(Q) 195 T (KR)(FMWH)(Q)(LPI) 196 E (FW)(VCAHG)(R)(Y) 124 T (R)(K)(H)(FW) 127 T (R)(K)(H)(FW) 133 Y (K)(QR)(E)(M) 212 D (R)(FWH)(YVCAG)(TK) 193 G (KER)(HD)(Q)(FMW) 166 G (R)(KE)(H)(FW) Fig. 4. A possible active surface on the chain 1iu9A. The larger cluster it 147 P (YR)(H)(T)(KE) belongs to is shown in blue. 126 G (R)(KE)(H)(D) 164 K (Y)(FW)(T)(H) Table 2. continued 160 Y (K)(Q)(M)(E) res type substitutions(%) cvg 163 V (Y)(R)(E)(K) 195 T T(98).S(1) 0.02 132 V (R)(KE)(Y)(D) 196 E E(96).D(2)H 0.03 198 S (R)(K)(H)(FW) 124 T T(93)AS(4)NE 0.04 200 V (R)(K)(E)(Y) 127 T T(93)L(1)CS(3)N 0.05 128 I (Y)(R)(H)(T) A(1) 133 Y Y(93)I(1)F(5)L 0.06 Table 3. Disruptive mutations for the surface patch in 1iu9A. 212 D D(85).(12)EN(1) 0.07 193 G A(6)G(91).(1) 0.08 166 G G(87)TD(1)VQ(1) 0.11 3 NOTES ON USING TRACE RESULTS HE(1)K(2)N(2)L. A(1) 3.1 Coverage 147 P P(86)SL(3)IT(2) 0.14 Trace results are commonly expressed in terms of coverage: the resi- HG(2)F(1)EKVAQ due is important if its “coverage” is small - that is if it belongs to 126 G G(35)F(24)Y(19) 0.15 some small top percentage of residues [100% is all of the residues NA(12)Q(4)T(1) in a chain], according to trace. The ET results are presented in the P(1)I form of a table, usually limited to top 25% percent of residues (or 164 K K(44)C(34)Q(1) 0.16 to some nearby percentage), sorted by the strength of the presumed S(5)A(3)V(4)G evolutionary pressure. (I.e., the smaller the coverage, the stronger the I(2)N(1)T(1)E.H pressure on the residue.) Starting from the top of that list, mutating a 160 Y Y(70)F(14)I(2) 0.17 couple of residues should affect the protein somehow, with the exact L(4).(1)C(1)SVH effects to be determined experimentally. R(2)EK 163 V V(26)L(56)K(3) 0.18 3.2 Known substitutions I(10)AY(2).Q One of the table columns is “substitutions” - other amino acid types 132 V V(14)F(49)KI(8) 0.19 seen at the same position in the alignment. These amino acid types M(3)L(19)W(1) may be interchangeable at that position in the protein, so if one wants A(1)Y(1)T to affect the protein by a point mutation, they should be avoided. For 198 S S(20)A(9)P(38). 0.21 example if the substitutions are “RVK” and the original protein has C(3)G(13)T(5) an R at that position, it is advisable to try anything, but RVK. Conver- M(4)N(1)REH(1) sely, when looking for substitutions which will not affect the protein, 200 V L(55).F(1)I(12) 0.24 one may try replacing, R with K, or (perhaps more surprisingly), with A(16)V(10)DG(1) V. The percentage of times the substitution appears in the alignment SYT is given in the immediately following bracket. No percentage is given 128 I V(8)M(52)I(12) 0.25 in the cases when it is smaller than 1%. This is meant to be a rough R(1)QL(13)K(2)T guide - due to rounding errors these percentages often do not add up Y(5)N(1) to 100%. 3.3 Surface Table 2. Residues forming surface ”patch” in 1iu9A. 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

3 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. 5. 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. 5. 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:

4 http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) http://mammoth.bcm.tmc.edu/traceview/ ”Protein structure alignment by incremental combinatorial extension The viewer is self-unpacking and self-installing. Input files to be used (CE) of the optimal path . Protein Engineering 11(9) 739-747. with ETV (extension .etvx) can be found in the attachment to the 4.3.3 DSSP In this work a residue is considered solvent accessi- main report. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, which is roughly the area needed for one water molecule to come in 4.5 Citing this work the contact with the residue. DSSP is copyrighted by W. Kabsch, C. The method used to rank residues and make predictions in this report Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of by [email protected] November 18,2002, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues by Importance” J. Mol. Bio. 336: 1265-82. For the original version http://www.cmbi.kun.nl/gv/dssp/descrip.html. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 4.3.4 HSSP Whenever available, report maker uses HSSP ali- tionary Trace Method Defines Binding Surfaces Common to Protein gnment as a starting point for the analysis (sequences shorter than Families” J. Mol. Bio. 257: 342-358. 75% of the query are taken out, however); R. Schneider, A. de report maker itself is described in Mihalek I., I. Res and O. Daruvar, and C. Sander. ”The HSSP database of protein structure- Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. of service for comparative analysis of proteins.” Bioinformatics 22:1656-7. http://swift.cmbi.kun.nl/swift/hssp/ 4.6 About report maker report maker was written in 2006 by Ivana Mihalek. The 1D ran- 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- king visualization program was written by Ivica Res.ˇ report maker Wesley,” Reading, Mass. (1986). is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), 4.7 Attachments ”MUSCLE: multiple sequence alignment with high accuracy and The following files should accompany this report: high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1iu9A.complex.pdb - coordinates of 1iu9A with all of its inter- http://www.drive5.com/muscle/ acting partners • 4.3.7 Pymol The figures in this report were produced using 1iu9A.etvx - ET viewer input file for 1iu9A Pymol. The scripts can be found in the attachment. Pymol • 1iu9A.cluster report.summary - Cluster report summary for is an open-source application copyrighted by DeLano Scien- 1iu9A tific LLC (2005). For more information about Pymol see • 1iu9A.ranks - Ranks file in sequence order for 1iu9A http://pymol.sourceforge.net/. (Note for Windows • 1iu9A.clusters - Cluster descriptions for 1iu9A users: the attached package needs to be unzipped for Pymol to read • the scripts and launch the viewer.) 1iu9A.msf - the multiple sequence alignment used for the chain 1iu9A 4.4 Note about ET Viewer • 1iu9A.descr - description of sequences used in 1iu9A msf Dan Morgan from the Lichtarge lab has developed a visualization • 1iu9A.ranks sorted - full listing of residues and their ranking for tool specifically for viewing trace results. If you are interested, please 1iu9A visit:

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