1Dwo Lichtarge Lab 2006

Pages 1–7 1dwo Evolutionary trace report by report maker April 9, 2010

4.3.1 Alistat 6 4.3.2 CE 6 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 1dwo): Title: Crystal structure of hydroxynitrile lyase from manihot esculenta in complex with substrates acetone and chloroace- tone:implications for the mechanism of cyanogenesis Compound: Mol id: 1; molecule: hydroxynitrile lyase; chain: a, b; synonym: (s)-acetone-cyanohydrin lyase, (s)- hydroxynitrilase; ec: 4.2.1.37; engineered: yes; other details: acetone complex Organism, scientiﬁc name: Manihot Esculenta; CONTENTS 1dwo contains a single unique chain 1dwoA (262 residues long) and its homologue 1dwoB. 1 Introduction 1

2 Chain 1dwoA 1 2 CHAIN 1DWOA 2.1 P52705 overview 1 2.1 P52705 overview 2.2 Multiple sequence alignment for 1dwoA 1 2.3 Residue ranking in 1dwoA 1 From SwissProt, id P52705, 94% identical to 1dwoA: 2.4 Top ranking residues in 1dwoA and their position on Description: (S)-acetone-cyanohydrin lyase (EC 4.1.2.39) ((S)- the structure 1 hydroxynitrile lyase) ((S)-hydroxynitrilase) (Oxynitrilase). 2.4.1 Clustering of residues at 25% coverage. 2 Organism, scientiﬁc name: Manihot esculenta (Cassava) (Manioc). 2.4.2 Overlap with known functional surfaces at Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 25% coverage. 2 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 2.4.3 Possible novel functional surfaces at 25% eudicotyledons; rosids; eurosids I; Malpighiales; Euphorbiaceae; coverage. 4 Crotonoideae; Manihoteae; Manihot. Function: Involved in cyanogenesis, the release of HCN from inju- 3 Notes on using trace results 5 red tissues. Decomposes a varieties of (R) or (S) cyanohydrins into 3.1 Coverage 5 HCN and the corresponding aldehydes and ketones. The natural 3.2 Known substitutions 5 substrate of this enzyme is (S)-acetone cyanohydrin. 3.3 Surface 5 Catalytic activity: 2-hydroxyisobutyronitrile = cyanide + acetone. 3.4 Number of contacts 6 Subunit: Homotrimer. 3.5 Annotation 6 Similarity: Belongs to the AB hydrolase superfamily. Hydroxynitrile 3.6 Mutation suggestions 6 lyase family. About: This Swiss-Prot entry is copyright. It is produced through a 4 Appendix 6 collaboration between the Swiss Institute of Bioinformatics and the 4.1 File formats 6 EMBL outstation - the European Bioinformatics Institute. There are 4.2 Color schemes used 6 no restrictions on its use as long as its content is in no way modiﬁed 4.3 Credits 6 and this statement is not removed.

1 Lichtarge lab 2006 2.4 Top ranking residues in 1dwoA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1dwoA colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this ﬁgure can be found in the attachment.

Fig. 1. Residues -4-127 in 1dwoA colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

Fig. 2. Residues 128-258 in 1dwoA colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

2.2 Multiple sequence alignment for 1dwoA For the chain 1dwoA, the alignment 1dwoA.msf (attached) with 33 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 1dwoA.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 1dwoA, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 33 Total number of residues: 8134 Smallest: 117 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 262 top 25% of all residues, this time colored according to clusters they Average length: 246.5 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 262 in Table 1. Average identity: 43% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 10% color residues Most distant seq: 40% red 62 6,7,8,9,10,11,14,15,16,17,19 26,27,30,31,35,37,40,42,57 Furthermore, <1% of residues show as conserved in this ali- 60,61,76,77,78,79,80,82,83 gnment. 87,89,93,96,101,102,105,107 The alignment consists of 96% eukaryotic ( 96% plantae) 108,162,166,169,173,175,190 sequences. (Descriptions of some sequences were not readily availa- 195,196,197,200,208,216,220 ble.) The ﬁle containing the sequence descriptions can be found in 224,228,235,236,239,241,243 the attachment, under the name 1dwoA.descr. 250,253,254,257

Table 1. Clusters of top ranking residues in 1dwoA. 2.3 Residue ranking in 1dwoA The 1dwoA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues 2.4.2 Overlap with known functional surfaces at 25% coverage. in 1dwoA can be found in the file called 1dwoA.ranks sorted in the The name of the ligand is composed of the source PDB identifier attachment. and the heteroatom name used in that file.

2 Fig. 4. Residues in 1dwoA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached.

Acetone binding site. Table 2 lists the top 25% of residues at the interface with 1dwoSACN259 (acetone). 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 antn (%) bb (A˚ ) 236 H H(96) 0.02 2/0 4.38 site .(3) 11 T G(75) 0.08 9/0 2.90 T(18) .(3) N(3) 80 S S(87) 0.12 9/2 3.01 site A(3) .(6) D(3) 14 H H(84) 0.15 3/0 4.15 I(3) L(6) .(3) F(3)

Table 2. The top 25% of residues in 1dwoA at the interface with acetone.(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˚ ) 236 H (E)(TQMD)(SNVCLAPIG)(K) .(3) 11 T (R)(K)(FWH)(M) V(3) 80 S (R)(K)(H)(FQW) 19 W W(90) 0.06 8/7 3.79 14 H (E)(T)(Q)(D) Y(6) .(3) Table 3. List of disruptive mutations for the top 25% of residues in 42 G G(93) 0.07 3/3 3.90 1dwoA, that are at the interface with acetone. .(6) 16 A A(81) 0.09 13/9 3.19 G(15) .(3) 37 D D(87) 0.14 1/0 4.45 .(6) E(3) N(3) 166 E D(87) 0.14 2/1 4.69 .(3) E(9) 35 A A(84) 0.19 1/0 3.76 V(9) .(6) 173 V L(84) 0.21 21/14 3.62 .(3) V(6) S(6) 27 E R(36) 0.23 3/0 4.02 E(51) .(6) K(3) V(3)

Table 4. The top 25% of residues in 1dwoA at the interface with 1dwoB. (Field names: res: residue number in the PDB entry; type: amino acid type; Fig. 5. Residues in 1dwoA, at the interface with acetone, colored by their substs: substitutions seen in the alignment; with the percentage of each type relative importance. The ligand (acetone) is colored green. Atoms further than in the bracket; noc/bb: number of contacts with the ligand, with the number of 30A˚ away from the geometric center of the ligand, as well as on the line of contacts realized through backbone atoms given in the bracket; dist: distance sight to the ligand were removed. (See Appendix for the coloring scheme for of closest apporach to the ligand. ) the protein chain 1dwoA.)

Figure 5 shows residues in 1dwoA colored by their importance, at the interface with 1dwoSACN259. Table 5. Interface with 1dwoB.Table 4 lists the top 25% of residues at res type disruptive the interface with 1dwoB. The following table (Table 5) suggests mutations possible disruptive replacements for these residues (see Section 3.6). 17 W (KE)(TQD)(SNCG)(R) 175 R (T)(D)(Y)(VCAG) Table 4. 169 L (Y)(R)(H)(T) res type subst’s cvg noc/ dist 19 W (K)(E)(Q)(D) (%) bb (A˚ ) 42 G (KER)(FQMWHD)(NLPI)(Y) 17 W W(96) 0.01 17/11 3.68 16 A (KER)(Y)(HD)(Q) .(3) 37 D (R)(FWH)(YVCAG)(TK) 175 R R(93) 0.03 10/1 3.88 166 E (FW)(H)(VCAG)(R) .(3) 35 A (YE)(KR)(D)(H) K(3) 173 V (R)(KY)(E)(H) 169 L L(93) 0.05 34/8 3.72 27 E (FW)(YH)(CG)(VA) continued in next column Table 5. List of disruptive mutations for the top 25% of residues in 1dwoA, that are at the interface with 1dwoB.

4 Fig. 6. Residues in 1dwoA, at the interface with 1dwoB, colored by their rela- Fig. 7. Residues in 1dwoA, at the interface with 1dwoA1, colored by their tive importance. 1dwoB is shown in backbone representation (See Appendix relative importance. 1dwoA1 is shown in backbone representation (See for the coloring scheme for the protein chain 1dwoA.) Appendix for the coloring scheme for the protein chain 1dwoA.)

Figure 6 shows residues in 1dwoA colored by their importance, at the 2.4.3 Possible novel functional surfaces at 25% coverage. One interface with 1dwoB. group of residues is conserved on the 1dwoA surface, away from (or Interface with 1dwoA1.By analogy with 1dwoB – 1dwoA1 inter- susbtantially larger than) other functional sites and interfaces reco- face. Table 6 lists the top 25% of residues at the interface with gnizable in PDB entry 1dwo. It is shown in Fig. 8. The right panel 1dwoA1. The following table (Table 7) suggests possible disruptive shows (in blue) the rest of the larger cluster this surface belongs to. replacements for these residues (see Section 3.6). Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) 93 Y F(72) 0.22 16/0 3.29 Y(18) L(6) .(3)

Table 6. The top 25% of residues in 1dwoA at the interface with 1dwoA1. (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 Fig. 8. A possible active surface on the chain 1dwoA. The larger cluster it of closest apporach to the ligand. ) belongs to is shown in blue.

Table 7. The residues belonging to this surface ”patch” are listed in Table res type disruptive 8, while Table 9 suggests possible disruptive replacements for these mutations residues (see Section 3.6). 93 Y (K)(Q)(EMR)(N) Table 8. res type substitutions(%) cvg antn Table 7. List of disruptive mutations for the top 25% of residues in 17 W W(96).(3) 0.01 1dwoA, that are at the interface with 1dwoA1. 236 H H(96).(3) 0.02 site 175 R R(93).(3)K(3) 0.03 Figure 7 shows residues in 1dwoA colored by their importance, at the continued in next column interface with 1dwoA1.

5 Table 8. continued to some nearby percentage), sorted by the strength of the presumed res type substitutions(%) cvg antn evolutionary pressure. (I.e., the smaller the coverage, the stronger the 243 T P(81).(3)T(15) 0.04 pressure on the residue.) Starting from the top of that list, mutating a 169 L L(93).(3)V(3) 0.05 couple of residues should affect the protein somehow, with the exact 42 G G(93).(6) 0.07 effects to be determined experimentally. 60 P P(93).(6) 0.07 11 T G(75)T(18).(3) 0.08 3.2 Known substitutions N(3) One of the table columns is “substitutions” - other amino acid types 16 A A(81)G(15).(3) 0.09 seen at the same position in the alignment. These amino acid types 80 S S(87)A(3).(6) 0.12 site may be interchangeable at that position in the protein, so if one wants D(3) to affect the protein by a point mutation, they should be avoided. For 37 D D(87).(6)E(3) 0.14 example if the substitutions are “RVK” and the original protein has N(3) an R at that position, it is advisable to try anything, but RVK. Conver- 166 E D(87).(3)E(9) 0.14 sely, when looking for substitutions which will not affect the protein, 162 C S(63)C(33).(3) 0.18 one may try replacing, R with K, or (perhaps more surprisingly), with 35 A A(84)V(9).(6) 0.19 V. The percentage of times the substitution appears in the alignment 57 Y Y(87)F(3)H(3) 0.21 is given in the immediately following bracket. No percentage is given .(6) in the cases when it is smaller than 1%. This is meant to be a rough 173 V L(84).(3)V(6) 0.21 guide - due to rounding errors these percentages often do not add up S(6) to 100%. 241 T S(69).(3)C(12) 0.24 T(15) 3.3 Surface To detect candidates for novel functional interfaces, first we look for Table 8. Residues forming surface ”patch” in 1dwoA. residues that are solvent accessible (according to DSSP program) by 2 at least 10A˚ , which is roughly the area needed for one water molecule to come in the contact with the residue. Furthermore, we require Table 9. that these residues form a “cluster” of residues which have neighbor ˚ res type disruptive within 5A from any of their heavy atoms. mutations Note, however, that, if our picture of protein evolution is correct, 17 W (KE)(TQD)(SNCG)(R) the neighboring residues which are not surface accessible might be 236 H (E)(TQMD)(SNVCLAPIG)(K) equally important in maintaining the interaction specificity - they 175 R (T)(D)(Y)(VCAG) should not be automatically dropped from consideration when choo- 243 T (R)(K)(H)(FW) sing the set for mutagenesis. (Especially if they form a cluster with 169 L (Y)(R)(H)(T) the surface residues.) 42 G (KER)(FQMWHD)(NLPI)(Y) 3.4 Number of contacts 60 P (YR)(TH)(SCG)(KE) 11 T (R)(K)(FWH)(M) Another column worth noting is denoted “noc/bb”; it tells the num- 16 A (KER)(Y)(HD)(Q) ber of contacts heavy atoms of the residue in question make across 80 S (R)(K)(H)(FQW) the interface, as well as how many of them are realized through the 37 D (R)(FWH)(YVCAG)(TK) backbone atoms (if all or most contacts are through the backbone, 166 E (FW)(H)(VCAG)(R) mutation presumably won’t have strong impact). Two heavy atoms ˚ 162 C (KR)(E)(FMWH)(Q) are considered to be “in contact” if their centers are closer than 5A. 35 A (YE)(KR)(D)(H) 3.5 Annotation 57 Y (K)(Q)(M)(E) 173 V (R)(KY)(E)(H) If the residue annotation is available (either from the pdb file or 241 T (KR)(FQMWH)(NELPI)(D) 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 9. Disruptive mutations for the surface patch in 1dwoA. 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],

6 by descending size): red, blue, yellow, green, purple, azure, tur- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, DarkOliveGreen, CornﬂowerBlue, grey55, burlywood, LimeGreen, COVERAGE tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated

V evolutionary pressure they experience can be seen in Fig. 9. 100% 50% 30% 5% 4.3 Credits 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These statistics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the V alignment length (e.g. including gap characters). Also shown are RELATIVE IMPORTANCE some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two Fig. 9. Coloring scheme used to color residues by their relative importance. sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant long aliphatic chain [EKRQM], OH-group possession [SDETY ], seq” is calculated by finding the maximum pairwise identity (best and NH2 group possession [NQRK]. The suggestions are listed relative) for all N sequences, then finding the minimum of these N according to how different they appear to be from the original amino numbers (hence, the most outlying sequence). alistat is copyrighted acid, and they are grouped in round brackets if they appear equally by HHMI/Washington University School of Medicine, 1992-2001, disruptive. From left to right, each bracketed group of amino acid and freely distributed under the GNU General Public License. types resembles more strongly the original (i.e. is, presumably, less 4.3.2 CE To map ligand binding sites from different disruptive) These suggestions are tentative - they might prove disrup- source structures, report maker uses the CE program: tive to the fold rather than to the interaction. Many researcher will http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) choose, however, the straightforward alanine mutations, especially in ”Protein structure alignment by incremental combinatorial extension the beginning stages of their investigation. (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A˚ 2, 4.1 File formats which is roughly the area needed for one water molecule to come in Files with extension “ranks sorted” are the actual trace results. The the contact with the residue. DSSP is copyrighted by W. Kabsch, C. fields in the table in this file: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 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 • 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 • rho ET score - the smaller this value, the lesser variability of 4.3.5 LaTex The text for this report was processed using LATEX; this position across the branches of the tree (and, presumably, Leslie Lamport, “LaTeX: A Document Preparation System Addison- the greater the importance for the protein) Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which 4.3.6 Muscle When making alignments “from scratch”, report have this rho or smaller maker uses Muscle alignment program: Edgar, Robert C. (2004), • gaps percentage of gaps in this column ”MUSCLE: multiple sequence alignment with high accuracy and 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 by cluster size: black is a single-residue cluster; clusters composed of 4.3.7 Pymol The figures in this report were produced using more than one residue colored according to this hierarchy (ordered Pymol. The scripts can be found in the attachment. Pymol

7 is an open-source application copyrighted by DeLano Scien- 4.6 About report maker tific LLC (2005). For more information about Pymol see report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://pymol.sourceforge.net/. (Note for Windows king visualization program was written by Ivica Res.ˇ report maker users: the attached package needs to be unzipped for Pymol to read is copyrighted by Lichtarge Lab, Baylor College of Medicine, the scripts and launch the viewer.) Houston. 4.4 Note about ET Viewer 4.7 Attachments Dan Morgan from the Lichtarge lab has developed a visualization The following files should accompany this report: tool specifically for viewing trace results. If you are interested, please • 1dwoA.complex.pdb - coordinates of 1dwoA with all of its visit: interacting partners http://mammoth.bcm.tmc.edu/traceview/ • 1dwoA.etvx - ET viewer input file for 1dwoA • 1dwoA.cluster report.summary - Cluster report summary for The viewer is self-unpacking and self-installing. Input files to be used 1dwoA with ETV (extension .etvx) can be found in the attachment to the • 1dwoA.ranks - Ranks file in sequence order for 1dwoA main report. • 1dwoA.clusters - Cluster descriptions for 1dwoA 4.5 Citing this work • 1dwoA.msf - the multiple sequence alignment used for the chain The method used to rank residues and make predictions in this report 1dwoA can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1dwoA.descr - description of sequences used in 1dwoA msf Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1dwoA.ranks sorted - full listing of residues and their ranking by Importance” J. Mol. Bio. 336: 1265-82. For the original version for 1dwoA of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1dwoA.1dwoSACN259.if.pml - Pymol script for Figure 5 tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. • 1dwoA.cbcvg - used by other 1dwoA – related pymol scripts report maker itself is described in Mihalek I., I. Res and O. • 1dwoA.1dwoB.if.pml - Pymol script for Figure 6 Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1dwoA.1dwoA1.if.pml - Pymol script for Figure 7 of service for comparative analysis of proteins.” Bioinformatics 22:1656-7.