Pages 1–5 3dlx Evolutionary trace report by report maker September 4, 2009

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 3dlx): Title: Crystal structure of human 3-oxoacid coa transferase 1 Compound: Mol id: 1; molecule: succinyl-coa:3-ketoacid- coenzyme a transferase 1; chain: a, b, c, d; synonym: somatic- type succinyl coa:3-oxoacid coa- transferase, scot-s; ec: 2.8.3.5; engineered: yes Organism, scientific name: Homo Sapiens; 3dlx contains a single unique chain 3dlxA (470 residues long) and its homologues 3dlxD, 3dlxC, and 3dlxB.

2 CHAIN 3DLXA 2.1 P55809 overview CONTENTS From SwissProt, id P55809, 95% identical to 3dlxA: Description: Succinyl-CoA:3-ketoacid-coenzyme A transferase 1, 1 Introduction 1 mitochondrial precursor (EC 2.8.3.5) (Somatic-type succinyl CoA:3- 2 Chain 3dlxA 1 oxoacid CoA- transferase) (Scot-S). 2.1 P55809 overview 1 Organism, scientific name: Homo sapiens (Human). 2.2 Multiple sequence alignment for 3dlxA 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.3 Residue ranking in 3dlxA 1 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.4 Top ranking residues in 3dlxA and their position on Catarrhini; Hominidae; Homo. the structure 2 Function: Key enzyme for ketone body catabolism. Transfers 2.4.1 Clustering of residues at 100% coverage. 2 the CoA moiety from succinate to acetoacetate. Formation of the enzyme-CoA intermediate proceeds via an unstable anhydride spe- 3 Notes on using trace results 3 cies formed between the carboxylate groups of the enzyme and 3.1 Coverage 3 substrate. 3.2 Known substitutions 3 Catalytic activity: Succinyl-CoA + a 3-oxo acid = succinate + a 3- 3.3 Surface 3 oxoacyl-CoA. 3.4 Number of contacts 3 Pathway: Ketolysis; first step. 3.5 Annotation 4 Subunit: Homodimer. 3.6 Mutation suggestions 4 Subcellular location: Mitochondrial matrix. Tissue specificity: Abundant in heart, followed in order by kidney, 4 Appendix 4 brain, and muscle, whereas in liver it is undetectable; also detectable 4.1 File formats 4 in leukocytes and fibroblasts. 4.2 Color schemes used 4 Disease: Defects in OXCT1 are a cause of ketoacidosis 4.3 Credits 4 [MIM:245050]. 4.3.1 Alistat 4 Similarity: Belongs to the 3-oxoacid CoA-transferase family. 4.3.2 CE 4 About: This Swiss-Prot entry is copyright. It is produced through a 4.3.3 DSSP 4 collaboration between the Swiss Institute of Bioinformatics and the 4.3.4 HSSP 4 EMBL outstation - the European Bioinformatics Institute. There are

1 Lichtarge lab 2006 Fig. 1. Residues 40-274 in 3dlxA colored by their relative importance. (See Fig. 2. Residues 275-519 in 3dlxA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.) Appendix, Fig.5, for the coloring scheme.) no restrictions on its use as long as its content is in no way modified get to 25%). Figure 3 shows residues in 3dlxA colored by their and this statement is not removed. importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for 2.2 Multiple sequence alignment for 3dlxA producing this figure can be found in the attachment. For the chain 3dlxA, the alignment 3dlxA.msf (attached) with 969 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 3dlxA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 969 Total number of residues: 228639 Smallest: 118 Largest: 470 Average length: 236.0 Alignment length: 470 Average identity: 30% Most related pair: 99% Most unrelated pair: 0% Most distant seq: 46%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 2% eukaryotic ( <1% vertebrata, <1% arthropoda, 1% fungi), 14% prokaryotic, and <1% archaean sequences. (Descriptions of some sequences were not readily availa- Fig. 3. Residues in 3dlxA, colored by their relative importance. Clockwise: ble.) The file containing the sequence descriptions can be found in front, back, top and bottom views. the attachment, under the name 3dlxA.descr.

2.3 Residue ranking in 3dlxA 2.4.1 Clustering of residues at 100% coverage. Fig. 4 shows the The 3dlxA sequence is shown in Figs. 1–2, with each residue colored top 100% of all residues, this time colored according to clusters they according to its estimated importance. The full listing of residues belong to. The clusters in Fig.4 are composed of the residues listed in 3dlxA can be found in the file called 3dlxA.ranks sorted in the in Table 1. attachment. 2.4 Top ranking residues in 3dlxA and their position on the structure In the following we consider residues ranking among top 100% of residues in the protein (the closest this analysis allows us to

2 Table 1. continued cluster size member color residues 214,215,216,217,218,219,220 221,222,223,224,225,226,227 228,229,230,231,232,233,234 235,236,237,238,239,240,241 242,243,244,245,246,247,248 249,250,251,252,253,254,255 256,257,258,259,260,261,262 263,264,265,266,267,268,269 270,271,272,273,274,275,276 277,278,279,280,281,282,283 284,285,286,297,298,299,300 301,302,303,304,305,306,307 308,309,310,311,312,313,314 315,316,317,318,319,320,321 322,323,324,325,326,327,328 329,330,331,332,333,334,335 336,337,338,339,340,341,342 343,344,345,346,347,348,349 350,351,352,353,354,355,356 Fig. 4. Residues in 3dlxA, colored according to the cluster they belong to: 357,358,359,360,361,362,363 red, followed by blue and yellow are the largest clusters (see Appendix for 364,365,366,367,368,369,370 the coloring scheme). Clockwise: front, back, top and bottom views. The 371,372,373,374,375,376,377 corresponding Pymol script is attached. 378,379,380,381,382,383,384 385,386,387,388,389,390,391 392,393,394,395,396,397,398 Table 1. 399,400,401,402,403,404,405 cluster size member 406,407,408,409,410,411,412 color residues 413,414,415,416,417,418,419 red 470 40,41,42,43,44,45,46,47,48 420,421,422,423,424,425,426 49,50,51,52,53,54,55,56,57 427,428,429,430,431,432,433 58,59,60,61,62,63,64,65,66 434,435,436,437,438,439,440 67,68,69,70,71,72,73,74,75 441,442,443,444,445,446,447 76,77,78,79,80,81,82,83,84 448,449,450,451,452,453,454 85,86,87,88,89,90,91,92,93 455,456,457,458,459,460,461 94,95,96,97,98,99,100,101 462,463,464,465,466,467,468 102,103,104,105,106,107,108 469,470,471,472,473,474,475 109,110,111,112,113,114,115 476,477,478,479,480,481,482 116,117,118,119,120,121,122 483,484,485,486,487,488,489 123,124,125,126,127,128,129 490,491,492,493,494,495,496 130,131,132,133,134,135,136 497,498,499,500,501,502,503 137,138,139,140,141,142,143 504,505,506,507,508,509,510 144,145,146,147,148,149,150 511,512,513,514,515,516,517 151,152,153,154,155,156,157 518,519 158,159,160,161,162,163,164 165,166,167,168,169,170,171 Table 1. 172,173,174,175,176,177,178 Clusters of top ranking residues in 3dlxA. 179,180,181,182,183,184,185 186,187,188,189,190,191,192 193,194,195,196,197,198,199 200,201,202,203,204,205,206 3 NOTES ON USING TRACE RESULTS 207,208,209,210,211,212,213 3.1 Coverage continued in next column 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 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

3 to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the pressure on the residue.) Starting from the top of that list, mutating a couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. COVERAGE 3.2 Known substitutions V

One of the table columns is “substitutions” - other amino acid types 100% 50% 30% 5% 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 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, V one may try replacing, R with K, or (perhaps more surprisingly), with RELATIVE IMPORTANCE V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given in the cases when it is smaller than 1%. This is meant to be a rough Fig. 5. Coloring scheme used to color residues by their relative importance. guide - due to rounding errors these percentages often do not add up to 100%. long aliphatic chain [EKRQM], OH-group possession [SDETY ], 3.3 Surface and NH2 group possession [NQRK]. The suggestions are listed To detect candidates for novel functional interfaces, first we look for according to how different they appear to be from the original amino residues that are solvent accessible (according to DSSP program) by 2 acid, and they are grouped in round brackets if they appear equally at least 10A˚ , which is roughly the area needed for one water mole- disruptive. From left to right, each bracketed group of amino acid cule to come in the contact with the residue. Furthermore, we require types resembles more strongly the original (i.e. is, presumably, less that these residues form a “cluster” of residues which have neighbor disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with 4 APPENDIX the surface residues.) 4.1 File formats 3.4 Number of contacts Files with extension “ranks sorted” are the actual trace results. The fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across • alignment# number of the position in the alignment the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, • residue# residue number in the PDB file mutation presumably won’t have strong impact). Two heavy atoms • type amino acid type ˚ are considered to be “in contact” if their centers are closer than 5A. • rank rank of the position according to older version of ET 3.5 Annotation • variability has two subfields: If the residue annotation is available (either from the pdb file or 1. number of different amino acids appearing in in this column from other sources), another column, with the header “annotation” of the alignment appears. Annotations carried over from PDB are the following: site 2. their type (indicating existence of related site record in PDB ), S-S (disulfide • rho ET score - the smaller this value, the lesser variability of bond forming residue), hb (hydrogen bond forming residue, jb (james this position across the branches of the tree (and, presumably, bond forming residue), and sb (for salt bridge forming residue). the greater the importance for the protein) • 3.6 Mutation suggestions cvg coverage - percentage of the residues on the structure which have this rho or smaller Mutation suggestions are completely heuristic and based on comple- • mentarity with the substitutions found in the alignment. Note that gaps percentage of gaps in this column they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following 4.2 Color schemes used properties: small [AV GSTC], medium [LPNQDEMIK], large The following color scheme is used in figures with residues colored [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- by cluster size: black is a single-residue cluster; clusters composed of tively [KHR], or negatively [DE] charged, aromatic [WFYH], more than one residue colored according to this hierarchy (ordered

4 by descending size): red, blue, yellow, green, purple, azure, tur- http://www.drive5.com/muscle/ quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, 4.3.7 Pymol The figures in this report were produced using bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, Pymol. The scripts can be found in the attachment. Pymol DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. is an open-source application copyrighted by DeLano Scien- The colors used to distinguish the residues by the estimated tific LLC (2005). For more information about Pymol see http://pymol.sourceforge.net/ evolutionary pressure they experience can be seen in Fig. 5. . (Note for Windows users: the attached package needs to be unzipped for Pymol to read 4.3 Credits the scripts and launch the viewer.) 4.3.1 Alistat alistat reads a multiple sequence alignment from the 4.4 Note about ET Viewer file and shows a number of simple statistics about it. These stati- Dan Morgan from the Lichtarge lab has developed a visualization stics include the format, the number of sequences, the total number tool specifically for viewing trace results. If you are interested, please of residues, the average and range of the sequence lengths, and the visit: alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- http://mammoth.bcm.tmc.edu/traceview/ ned as (idents / MIN(len1, len2)) where idents is the number of The viewer is self-unpacking and self-installing. Input files to be used exact identities and len1, len2 are the unaligned lengths of the two with ETV (extension .etvx) can be found in the attachment to the sequences. The ”average percent identity”, ”most related pair”, and main report. ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant 4.5 Citing this work seq” is calculated by finding the maximum pairwise identity (best The method used to rank residues and make predictions in this report relative) for all N sequences, then finding the minimum of these N can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of numbers (hence, the most outlying sequence). alistat is copyrighted Evolution-Entropy Hybrid Methods for Ranking of Protein Residues by HHMI/Washington University School of Medicine, 1992-2001, by Importance” J. Mol. Bio. 336: 1265-82. For the original version and freely distributed under the GNU General Public License. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 4.3.2 CE To map ligand binding sites from different tionary Trace Method Defines Binding Surfaces Common to Protein source structures, report maker uses the CE program: Families” J. Mol. Bio. 257: 342-358. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) report maker itself is described in Mihalek I., I. Res and O. ”Protein structure alignment by incremental combinatorial extension Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type (CE) of the optimal path . Protein Engineering 11(9) 739-747. of service for comparative analysis of proteins.” Bioinformatics 22:1656-7. 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, 4.6 About report maker which is roughly the area needed for one water molecule to come in report maker was written in 2006 by Ivana Mihalek. The 1D ran- the contact with the residue. DSSP is copyrighted by W. Kabsch, C. king visualization program was written by Ivica Res.ˇ report maker Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version is copyrighted by Lichtarge Lab, Baylor College of Medicine, by [email protected] November 18,2002, Houston. http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.7 Attachments The following files should accompany this report: 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • gnment as a starting point for the analysis (sequences shorter than 3dlxA.complex.pdb - coordinates of 3dlxA with all of its inter- 75% of the query are taken out, however); R. Schneider, A. de acting partners Daruvar, and C. Sander. ”The HSSP database of protein structure- • 3dlxA.etvx - ET viewer input file for 3dlxA sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 3dlxA.cluster report.summary - Cluster report summary for 3dlxA http://swift.cmbi.kun.nl/swift/hssp/ • 3dlxA.ranks - Ranks file in sequence order for 3dlxA 4.3.5 LaTex The text for this report was processed using LATEX; • 3dlxA.clusters - Cluster descriptions for 3dlxA Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 3dlxA.msf - the multiple sequence alignment used for the chain Wesley,” Reading, Mass. (1986). 3dlxA 4.3.6 Muscle When making alignments “from scratch”, report • 3dlxA.descr - description of sequences used in 3dlxA msf maker uses Muscle alignment program: Edgar, Robert C. (2004), • 3dlxA.ranks sorted - full listing of residues and their ranking for ”MUSCLE: multiple sequence alignment with high accuracy and 3dlxA high throughput.” Nucleic Acids Research 32(5), 1792-97.

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