1. Bioinformatics Analysis of YqjG: an introduction and some questions YqjG: “Uncharacterized protein” from Escherichia coli UniProt ID = P42620 (YQJG_ECOLI) GenBank GI = 1176792 General annotation from UniProt: Belongs to the GST superfamily. Omega family. Contains 1 GST C-terminal domain.

2. Theta GSTT1_HUMAN Omega GSTO1_HUMAN Zeta GSTA1_HUMAN Tau GSTU1_ORYSJ Phi GSTF1_MAIZE Sigma GST_OMMSL Pi GSTP_ONCVO Mu GSTM1_RAT Alpha GSTA3_CHICK Beta EGST_ECOLI Overall sequence similarity network containing 2,851 sequences and 97,144 edges. Edges represent BLAST e-values of 1e-18 or more significance. Large nodes are colored by the classification of the amino acid sequence in SWISS-PROT and indicate a representative member of each subgroup. Nu YGHU_ECOLI Close Up of the Cluster – 96 sequences YqjG in black  Escherichia coli K-12 328 amino acids, 37.4 kD Annotated as being part of the omega class Q8NR03 in pink  Corynebacterium glutamicum  3M1G structure Network Generated by S. Brown – August 2010

3. Structural Similarity to Omega Class GSTs Subunit 1 (teal, blue) : Subunit 2 (pink/red) Subunit 1 (green/lemon) : Subunit 2 (teal,blue) PDB Code 1EEM Omega class glutathione transferase from homo sapiens YqjG a Glutathione Transferase from Escherichia coli

4. Alpha Helix 3 Alpha Helix A Alpha Helix A PDB Code 1EEM Omega class glutathione transferase from homo sapiens Subunit 1 (teal, blue) : Subunit 2 (pink/red) Dimer Interface Formation in the Omega Class

5. Shared Mechanism of Dimer Interface Formation Throughout Many GST Classes Omega class (1EEM) Nu class (3GX0)Beta class (1N2A) Zeta class - MAII (1FW1) Alpha class - MAII (1VF1) Phi class (1BYE)

6. N C Beta 1 Alpha 1 Beta 2 Alpha 2 Beta 3 Beta 4 Alpha 3 ABCD Mechanism of Dimer Interface Formation in “Traditional” GST classes N-terminal thioredoxin domain Alpha helical domain

7. C-terminal loop C-terminal loop Loop from Alpha Helix C-D YqjG – Glutathione Transferase from Escherichia coli Subunit 1 (green/lemon) : Subunit 2 (teal,blue) Formation of the dimer interface C Beta 1 Alpha 1 Beta 2 Alpha 2 Beta 3 Beta 4 Alpha 3 All Alpha Helical Domain ABCD N

8. C-terminal loop C-terminal loop Loop from Alpha Helix C-D N C Beta 1 Alpha 1 Beta 2 Alpha 2 Beta 3 Beta 4 Alpha 3 All Alpha Helical Domain ABCD N Subunit 1 (lemon/orange) : Subunit 2 (magenta/pink) Formation of the dimer interface PDB Code 3M1G The structure of a putative glutathione S-transferase from Corynebacterium glutamicum Midwest Center for Structural Genomics, unpublished

9. Structural superposition of YqjG (colored) and 3M1G (grey)

10. Identical Conserved Semi-conserved Sequence Conservation Map 90°

11. Electrostatic Surface Map 90°

12. Electrostatic Surface MapSequence Conservation Map +/ /-/+- - YQJG_ECOLI VYKAGFATSQEAYDEAVAKVFESLARLEQILGQHRYLTGNQLTEADIRLWTTLVRFDPVYVTHFKCDKHRISDY Q8NR03_CORGL VYRTGFAGSQEAHNEAYKRLWVALDWLEDRLSTRRYLMGDHITEADIRLYPTLVRFDAVYHGHFKCGRNKITEM **::*** ****::** ::: :* **: *. :*** *:::*******:.******.** ****.:::*:: / / // // YQJG_ECOLI LNLYGFLRDIYQMPGIAETVNFDHIRNHYFRSHKTINPTGIISIGP-WQDLDEPHGRDVRFG-------------- Q8NR03_CORGL PNLWGYLRDLFQTPGFGDTTDFTEIKQHYYITHAEINPTRIVPVGPDLSGFATPHGREKLGGSPFAEGVTLPGPIP **:*:***::* **:.:*.:*.*::**: :* **** *:.:**..: ****: * E 204 D 160 K 158 K 161 R 163

13. A New “Lock and Key” Motif PDB Code 1EEM Omega class glutathione transferase. GST Classes Alpha/Mu/Phi/Omega Hydrophobic interface “Lock and Key” mechanism PDB Code 1XW6 Mu class glutathione transferase.

14. A New “Lock and Key” Motif: YqjG

15. CLUSTAL 2.0.12 multiple sequence alignment sp|P42620|YQJG_ECOLI MGQLIDGVWHDTWYDTKSTGGKFQRSASAFRNWLTADGAPG------PTG 44 tr|Q8NR03|Q8NR03_CORGL MANTSS-DWAGAPQN-ASADGEFVRDTNYIDDRIVADVPAGSEPIAQEDG 48 *.:. *.: : *:.*:* *.:. : : :.**..* * --β1-- -----α1------ -β2- sp|P42620|YQJG_ECOLI TGGFIAEKDRYHLYVSLACPWAHRTLIMRKLKGLEPFISVSVVNPLMLEN 94 tr|Q8NR03|Q8NR03_CORGL TFHWPVEAGRYRLVAARACPWAHRTVITRRLLGLENVISLGLTGPTHDVR 98 * :.*.**:*.: ********:* *:* ***.**:.:..*. ------α2------ -β3- -β4 sp|P42620|YQJG_ECOLI GWTFDDSFPGATGDTLYQNEFLYQLYLHADPHYSGRVTVPVLWDKKNHTI 144 tr|Q8NR03|Q8NR03_CORGL SWTFD--LDPNHLDPVLQIPRLQDAYFNRFPDYPRGITVPALVEESSKKV 146.**** : *.: * * : *:: *.*. :***.* ::..:.: -- -----α3-------- ---------αA------- sp|P42620|YQJG_ECOLI VSNESAEIIRMFNTAFDALG-AKAGDYYPPALQTKIDELNGWIYDTVNNG 193 tr|Q8NR03|Q8NR03_CORGL VTNDYPSITIDFNLEWKQFHREGAPNLYPAELREEMAPVMKRIFTEVNNG 196 *:*:..* ** :. : * : **. *: :: : *: **** ------ --K-------αB----------- -----αC- sp|P42620|YQJG_ECOLI VYKAGFATSQEAYDEAVAKVFESLARLEQILGQHRYLTGNQLTEADIRLW 243 tr|Q8NR03|Q8NR03_CORGL VYRTGFAGSQEAHNEAYKRLWVALDWLEDRLSTRRYLMGDHITEADIRLY 246 **::*** ****::** ::: :* **: *. :*** *:::*******: ------- -----αD------ sp|P42620|YQJG_ECOLI TTLVRFDPVYVTHFKCDKHRISDYLNLYGFLRDIYQMPGIAETVNFDHIR 293 tr|Q8NR03|Q8NR03_CORGL PTLVRFDAVYHGHFKCGRNKITEMPNLWGYLRDLFQTPGFGDTTDFTEIK 296.******.** ****.:::*:: **:*:***::* **:.:*.:*.*: L sp|P42620|YQJG_ECOLI NHYFRSHKTINPTGIISIGP-WQDLDEPHGRDVRFG-------------- 328 tr|Q8NR03|Q8NR03_CORGL QHYYITHAEINPTRIVPVGPDLSGFATPHGREKLGGSPFAEGVTLPGPIP 346 :**: :* **** *:.:**..: ****: * sp|P42620|YQJG_ECOLI ------------- tr|Q8NR03|Q8NR03_CORGL AGEEVKNPEPFQK 359 GSH Binding Dimer Interface Lock & Key

16. Preliminary bioinformatics analysis and some questions YqjG and related sequences form a unique cluster. - What about the sequences in the YqjG cluster make them different/distinct from all other glutathione transferases. There are two structures from this subset of sequences that have this new, unique dimer interface. - Will all sequences in this cluster have the same dimer interface? - What is the sequence conservation at the dimer interface in this subset of sequences? - How does this set of sequences differ from other GST classes at the dimer interface? 3M1G and YqjG separate from each other around e -75 but we know they have the same dimer interface, what is making them separate at these e-values.

17. 1e -14 1e -50 1e -75 1e -100 YqjG – Escherichia coli 3M1G – Corynebacterium glutamicum PcpF – Sphingobium chlorophenolicum

18. YqjG – Escherichia coli 3M1G – Corynebacterium glutamicum PcpF – Sphingobium chlorophenolicum 1e -50 1e -80 1e -100 1e -125

19. +/ /-/+- - YQJG_ECOLI VYKAGFATSQEAYDEAVAKVFESLARLEQILGQHRYLTGNQLTEADIRLWTTLVRFDPVYVTHFKCDKHRISDY Q8NR03_CORGL VYRTGFAGSQEAHNEAYKRLWVALDWLEDRLSTRRYLMGDHITEADIRLYPTLVRFDAVYHGHFKCGRNKITEM **::*** ****::** ::: :* **: *. :*** *:::*******:.******.** ****.:::*:: / / // // YQJG_ECOLI LNLYGFLRDIYQMPGIAETVNFDHIRNHYFRSHKTINPTGIISIGP-WQDLDEPHGRDVRFG-------------- Q8NR03_CORGL PNLWGYLRDLFQTPGFGDTTDFTEIKQHYYITHAEINPTRIVPVGPDLSGFATPHGREKLGGSPFAEGVTLPGPIP **:*:***::* **:.:*.:*.*::**: :* **** *:.:**..: ****: * Multiple sequence alignment of 95 sequences in the YqjG ‘cluster’

20. Multiple sequence alignment 95 sequences 68 sequences