Pages 1–6 1ej1 Evolutionary trace report by report maker September 18, 2008

4.3.3 DSSP 6 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 Data Bank entry (PDB id 1ej1): Title: Cocrystal structure of the messenger rna 5’ cap-binding protein () bound to 7-methyl-gdp Compound: Mol id: 1; molecule: eukaryotic translation initiation factor 4e; chain: a, b; fragment: residues 28-217; engineered: yes Organism, scientific name: Mus Musculus; 1ej1 contains a single unique chain 1ej1B (190 residues long) and its homologue 1ej1A.

CONTENTS 2 CHAIN 1EJ1B 2.1 P63074 overview 1 Introduction 1 From SwissProt, id P63074, 100% identical to 1ej1B: 2 Chain 1ej1B 1 Description: Eukaryotic translation initiation factor 4E (eIF4E) (eIF- 2.1 P63074 overview 1 4E) (mRNA cap-binding protein) (eIF-4F 25 kDa subunit). 2.2 Multiple sequence alignment for 1ej1B 1 Organism, scientific name: Rattus norvegicus (Rat). 2.3 Residue ranking in 1ej1B 2 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Verte- 2.4 Top ranking residues in 1ej1B and their position on brata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; the structure 2 Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Rattus. 2.4.1 Clustering of residues at 25% coverage. 2 Function: Recognizes and binds the 7-methylguanosine-containing 2.4.2 Overlap with known functional surfaces at mRNA cap during an early step in the initiation of protein synthesis 25% coverage. 2 and facilitates ribosome binding by inducing the unwinding of the mRNAs secondary structures. May play an important role in sperma- 3 Notes on using trace results 4 togenesis through translational regulation of stage- specific mRNAs 3.1 Coverage 4 during germ cell development. 3.2 Known substitutions 4 Subunit: eIF4F is a multi-subunit complex, the composition of 3.3 Surface 5 which varies with external and internal environmental conditions. 3.4 Number of contacts 5 It is composed of at least EIF4A, EIF4E and EIF4G1/EIF4G3. 3.5 Annotation 5 EIF4E is also known to interact with other partners. The interaction 3.6 Mutation suggestions 5 with EIF4ENIF1 mediates the import into the nucleus. Nonphos- phorylated EIF4EBP1, EIF4EBP2 and EIF4EBP3 compete with 4 Appendix 5 EIF4G1/EIF4G3 to interact with EIF4E; insulin stimulated MAP- 4.1 File formats 5 kinase (MAPK1 and MAPK3) phosphorylation of EIF4EBP1 causes 4.2 Color schemes used 5 dissociation of the complex allowing EIF4G1/EIF4G3 to bind and 4.3 Credits 5 consequent initiation of translation. Rapamycin can attenuate insulin 4.3.1 Alistat 5 stimulation, mediated by FKBPs. Interacts mutually exclusive with 4.3.2 CE 6 EIF4A1 AND EIF4A2 (By similarity).

1 Lichtarge lab 2006 2.4 Top ranking residues in 1ej1B and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 1ej1B 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 28-217 in 1ej1B colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Tissue specificity: Very high levels in post-meiotic testicular germ cells of rats of reproductive age. Ptm: Phosphorylation increases the ability of the protein to bind to mRNA caps and to form the eIF4F complex (By similarity). Similarity: Belongs to the eukaryotic initiation factor 4E family. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed.

2.2 Multiple sequence alignment for 1ej1B For the chain 1ej1B, the alignment 1ej1B.msf (attached) with 64 sequences was used. The alignment was assembled through combi- nation of BLAST searching on the UniProt database and alignment using Muscle program. It can be found in the attachment to this report, under the name of 1ej1B.msf. Its statistics, from the alistat Fig. 2. Residues in 1ej1B, colored by their relative importance. Clockwise: program are the following: front, back, top and bottom views.

Format: MSF Number of sequences: 64 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the Total number of residues: 11583 top 25% of all residues, this time colored according to clusters they Smallest: 144 belong to. The clusters in Fig.3 are composed of the residues listed Largest: 190 in Table 1. Average length: 181.0 Alignment length: 190 Table 1. Average identity: 37% cluster size member Most related pair: 99% color residues Most unrelated pair: 17% red 47 39,43,45,46,56,66,69,70,72 Most distant seq: 27% 73,76,82,85,90,91,94,95,98 100,102,103,104,107,110,111 112,113,127,130,131,134,135 Furthermore, 2% of residues show as conserved in this alignment. 139,140,142,150,151,153,157 The alignment consists of 98% eukaryotic ( 31% vertebrata, 10% 166,167,179,180,184,187,197 arthropoda, 20% fungi, 21% plantae) sequences. (Descriptions of 200 some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name Table 1. Clusters of top ranking residues in 1ej1B. 1ej1B.descr.

2.3 Residue ranking in 1ej1B 2.4.2 Overlap with known functional surfaces at 25% coverage. The 1ej1B sequence is shown in Fig. 1, with each residue colored The name of the ligand is composed of the source PDB identifier according to its estimated importance. The full listing of residues and the heteroatom name used in that file. in 1ej1B can be found in the file called 1ej1B.ranks sorted in the M7G binding site. Table 2 lists the top 25% of residues at the attachment. interface with 1ej1M7G1001 (m7g). The following table (Table 3)

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) Q(1) R(32) V(4) T(1) 101 M K(25) 0.25 12/8 3.05 M(46) E(12) A(3) L(4) V(3) I(1) S(1) T(1)

Table 2. The top 25% of residues in 1ej1B at the interface with M7G.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: Fig. 3. Residues in 1ej1B, colored according to the cluster they belong to: distance of closest apporach to the ligand. ) 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. Table 3. res type disruptive mutations suggests possible disruptive replacements for these residues (see 100 P (YR)(TH)(SKECG)(FQWD) Section 3.6). 166 W (KE)(TQD)(SNCRG)(M) 102 W (TE)(KD)(SCG)(QR) Table 2. 103 E (FWH)(R)(Y)(VA) res type subst’s cvg noc/ dist 157 R (T)(Y)(D)(SVCAG) ˚ (%) bb (A) 90 D (R)(FW)(H)(Y) 100 P P(100) 0.03 3/3 4.26 56 W (K)(E)(Q)(D) 166 W W(100) 0.03 4/0 3.78 112 R (YD)(T)(E)(FW) 102 W W(98) 0.05 98/8 2.67 101 M (Y)(H)(R)(T) Q(1) 103 E E(98) 0.05 19/1 2.67 Table 3. List of disruptive mutations for the top 25% of residues in 1ej1B, G(1) that are at the interface with M7G. 157 R R(93) 0.08 22/0 2.99 K(4) Q(1) Figure 4 shows residues in 1ej1B colored by their importance, at the 90 D D(84) 0.13 2/0 4.75 interface with 1ej1M7G1001. E(1) Interface with 1ej1A.Table 4 lists the top 25% of residues at the T(3) interface with 1ej1A. The following table (Table 5) suggests possible N(3) disruptive replacements for these residues (see Section 3.6). S(6) Table 4. R(1) res type subst’s cvg noc/ dist 56 W W(68) 0.15 104/0 3.20 (%) bb (A˚ ) Y(21) 73 W W(92) 0.09 46/0 3.19 .(3) L(4) C(4) .(1) F(1) F(1) 112 R K(59) 0.16 1/0 4.24 187 L V(3) 0.15 10/10 3.85 continued in next column L(84) I(6) continued in next column

3 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) 76 Y Y(79) 0.24 5/0 3.92 L(3) F(6) I(1) V(1) M(3) C(1) Q(3)

Table 4. The top 25% of residues in 1ej1B at the interface with 1ej1A. (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 5. res type disruptive Fig. 4. Residues in 1ej1B, at the interface with M7G, colored by their relative mutations importance. The ligand (M7G) is colored green. Atoms further than 30A˚ away 73 W (KE)(T)(QD)(SCRG) from the geometric center of the ligand, as well as on the line of sight to the 187 L (R)(Y)(T)(H) ligand were removed. (See Appendix for the coloring scheme for the protein 70 E (H)(FW)(Y)(R) chain 1ej1B.) 69 V (R)(Y)(KE)(H) 131 L (Y)(R)(T)(H) 135 L (R)(Y)(H)(TKE) Table 4. continued 76 Y (K)(R)(EQ)(M) res type subst’s cvg noc/ dist (%) bb (A˚ ) Table 5. .(4) List of disruptive mutations for the top 25% of residues in 1ej1B, that are at the interface with 1ej1A. F(1) 70 E E(85) 0.16 7/0 3.87 Q(6) Figure 5 shows residues in 1ej1B colored by their importance, at the P(4) interface with 1ej1A. .(1) S(1) 3 NOTES ON USING TRACE RESULTS 69 V V(87) 0.18 5/0 3.71 3.1 Coverage I(7) .(1) Trace results are commonly expressed in terms of coverage: the resi- A(1) due is important if its “coverage” is small - that is if it belongs to L(1) some small top percentage of residues [100% is all of the residues 131 L L(62) 0.18 3/0 3.45 in a chain], according to trace. The ET results are presented in the E(23) form of a table, usually limited to top 25% percent of residues (or D(1) to some nearby percentage), sorted by the strength of the presumed M(1) evolutionary pressure. (I.e., the smaller the coverage, the stronger the H(1) pressure on the residue.) Starting from the top of that list, mutating a K(6) couple of residues should affect the protein somehow, with the exact C(1) effects to be determined experimentally. F(1) 3.2 Known substitutions 135 L M(17) 0.19 8/0 3.65 L(78) One of the table columns is “substitutions” - other amino acid types A(1) seen at the same position in the alignment. These amino acid types F(1) may be interchangeable at that position in the protein, so if one wants C(1) to affect the protein by a point mutation, they should be avoided. For continued in next column 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,

4 appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue).

3.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following properties: small [AV GST C], medium [LP NQDEMIK], large [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- tively [KHR], or negatively [DE] charged, aromatic [W F Y H], long aliphatic chain [EKRQM], OH-group possession [SDET Y ], and NH2 group possession [NQRK]. The suggestions are listed according to how different they appear to be from the original amino acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will choose, however, the straightforward alanine mutations, especially in Fig. 5. Residues in 1ej1B, at the interface with 1ej1A, colored by their rela- the beginning stages of their investigation. tive importance. 1ej1A is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1ej1B.) 4 APPENDIX 4.1 File formats one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment Files with extension “ranks sorted” are the actual trace results. The is given in the immediately following bracket. No percentage is given fields in the table in this file: in the cases when it is smaller than 1%. This is meant to be a rough • alignment# guide - due to rounding errors these percentages often do not add up number of the position in the alignment to 100%. • residue# residue number in the PDB file • type amino acid type 3.3 Surface • rank rank of the position according to older version of ET To detect candidates for novel functional interfaces, first we look for • residues that are solvent accessible (according to DSSP program) by variability has two subfields: 2 at least 10A˚ , which is roughly the area needed for one water mole- 1. number of different amino acids appearing in in this column cule to come in the contact with the residue. Furthermore, we require of the alignment that these residues form a “cluster” of residues which have neighbor 2. their type within 5A˚ from any of their heavy atoms. • rho ET score - the smaller this value, the lesser variability of Note, however, that, if our picture of protein evolution is correct, this position across the branches of the tree (and, presumably, the neighboring residues which are not surface accessible might be the greater the importance for the protein) equally important in maintaining the interaction specificity - they • cvg coverage - percentage of the residues on the structure which should not be automatically dropped from consideration when choo- have this rho or smaller sing the set for mutagenesis. (Especially if they form a cluster with • the surface residues.) gaps percentage of gaps in this column 3.4 Number of contacts 4.2 Color schemes used Another column worth noting is denoted “noc/bb”; it tells the num- The following color scheme is used in figures with residues colored ber of contacts heavy atoms of the residue in question make across by cluster size: black is a single-residue cluster; clusters composed of the interface, as well as how many of them are realized through the more than one residue colored according to this hierarchy (ordered backbone atoms (if all or most contacts are through the backbone, by descending size): red, blue, yellow, green, purple, azure, tur- mutation presumably won’t have strong impact). Two heavy atoms quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, are considered to be “in contact” if their centers are closer than 5A˚ . bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, 3.5 Annotation tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. If the residue annotation is available (either from the pdb file or The colors used to distinguish the residues by the estimated from other sources), another column, with the header “annotation” evolutionary pressure they experience can be seen in Fig. 6.

5 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). 4.3.6 Muscle When making alignments “from scratch”, report COVERAGE maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and V high throughput.” Nucleic Acids Research 32(5), 1792-97. 100% 50% 30% 5% http://www.drive5.com/muscle/

4.3.7 Pymol The figures in this report were produced using Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien-

V tific LLC (2005). For more information about Pymol see http://pymol.sourceforge.net/. (Note for Windows RELATIVE IMPORTANCE users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) Fig. 6. Coloring scheme used to color residues by their relative importance. 4.4 Note about ET Viewer Dan Morgan from the Lichtarge lab has developed a visualization 4.3 Credits tool specifically for viewing trace results. If you are interested, please visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- http://mammoth.bcm.tmc.edu/traceview/ stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version seq” is calculated by finding the maximum pairwise identity (best of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- relative) for all N sequences, then finding the minimum of these N tionary Trace Method Defines Binding Surfaces Common to Protein numbers (hence, the most outlying sequence). alistat is copyrighted Families” J. Mol. Bio. 257: 342-358. by HHMI/Washington University School of Medicine, 1992-2001, report maker itself is described in Mihalek I., I. Res and O. and freely distributed under the GNU General Public License. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of .” Bioinformatics 4.3.2 CE To map ligand binding sites from different 22:1656-7. source structures, report maker uses the CE program: http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 4.6 About report maker ”Protein structure alignment by incremental combinatorial extension report maker was written in 2006 by Ivana Mihalek. The 1D ran- (CE) of the optimal path . Protein Engineering 11(9) 739-747. king visualization program was written by Ivica Res.ˇ report maker 4.3.3 DSSP In this work a residue is considered solvent accessi- is copyrighted by Lichtarge Lab, Baylor College of Medicine, 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , Houston. which is roughly the area needed for one water molecule to come in 4.7 Attachments the contact with the residue. DSSP is copyrighted by W. Kabsch, C. The following files should accompany this report: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, • 1ej1B.complex.pdb - coordinates of 1ej1B with all of its inter- acting partners http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1ej1B.etvx - ET viewer input file for 1ej1B 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • 1ej1B.cluster report.summary - Cluster report summary for gnment as a starting point for the analysis (sequences shorter than 1ej1B 75% of the query are taken out, however); R. Schneider, A. de • 1ej1B.ranks - Ranks file in sequence order for 1ej1B ”The HSSP database of protein structure- Daruvar, and C. Sander. • sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 1ej1B.clusters - Cluster descriptions for 1ej1B • 1ej1B.msf - the multiple sequence alignment used for the chain http://swift.cmbi.kun.nl/swift/hssp/ 1ej1B

6 • 1ej1B.descr - description of sequences used in 1ej1B msf • 1ej1B.cbcvg - used by other 1ej1B – related pymol scripts • 1ej1B.ranks sorted - full listing of residues and their ranking for • 1ej1B.1ej1A.if.pml - Pymol script for Figure 5 1ej1B • 1ej1B.1ej1M7G1001.if.pml - Pymol script for Figure 4

7