TIM BARREL Teresa Pramo Miguel Hernndez Diana Garzn

TIM BARREL Teresa Páramo Miguel Hernández Diana Garzón

Index n Introduction ¨ ¨ ¨ n n Super families Conserved regions ¨ ¨ ¨ n Comparison & prediction Evolution ¨ ¨ ¨ n Examples Residues Super families & functions Active sites ¨ n Structure Composition Loop regions Evolution of sequences Super imposition Gene fusion Fold change ¨ ¨ Insertion, Deletion & Substitution Circular permutations Strand Invasion Beta Hairpin Flip Swap

Introduction n The TIM barrel is an extremely common protein fold (which constitutes nearly 10% of all known enzymes) consisting of eight α -helices and eight parallel β-strands that alternate along the peptide backbone.

Introduction n Lineage ¨ Class: α/β Proteins ¨ Fold: TIM α/β Barrel ¨ Super families: n 32 super families

Structure n The α-helices and β-strands form a solenoid that curves around to close on itself in a doughnut shape, topologically known as a toroid.

Structure n The parallel β-strands form the inner wall of the doughnut (hence, a β-barrel), whereas the α-helices form the outer wall of the doughnut.

Composition n The protein's core is tightly packed with bulky hydrophobic amino acid residues. The packing interactions between the sheets and helices are also dominated by hydrophobicity. The branched aliphatic residues valine, leucine, and isoleucine comprise about 40% of the total residues in the β-strands.

Loop regions n n n Out of the 200 residues required to form a TIM barrel, only 160 are considered structurally equivalent between different proteins sharing this fold. The remaining residues are located on the loop regions that link the helices and sheets; the loops at the Cterminal end of the sheets tend to contain the active site, which is one reason this fold is so common. The residues required to maintain the structure and the residues that effect enzymatic catalysis are for the most part distinct subsets. The linking loops can, in fact, be so long that they contain other protein domains.

Super families 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Triosephosphate isomerase (TIM) Ribulose-phoshate binding barrel Thiamin phosphate synthase Pyridoxine 5'-phosphate synthase FMN-linked oxidoreductases Inosine monophosphate dehydrogenase (IMPDH) PLP-binding barrel NAD(P)-linked oxidoreductase (Trans)glycosidases Metallo-dependent hydrolases Aldolase Enolase C-terminal domain-like Phosphoenolpyruvate/pyruvate domain Malate synthase G Ru. Bis. Co, C-terminal domain Xylose isomerase-like Bacterial luciferase-like 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Nicotinate/Quinolinate PRTase Cterminal domain-like PLC-like phosphodiesterases Cobalamin (vitamin B 12)-dependent enzymes t. RNA-guanine transglycosylase Dihydropteroate synthetase-like UROD/Met. E-like FAD-linked oxidoreductase Monomethylamine methyltransferase Mtm. B Homocysteine S-methyltransferase (2 r)-phospho-3 -sulfolactate synthase Com. A Radical SAM enzymes Glp. P-like Cut. C-like Thi. G-like TM 1631 -like

Conserved regions n As structure in general is more conserved than sequence, thorough structural comparisons and structure-based sequence alignments can help, in cases where standard sequence-alignment tools fail, to determine distant evolutionary relationships. n One of the most intriguing features among members of this class of proteins is although they all exhibit the same tertiary fold there is very little sequence homology between them. n Of the approximately 200 residues required to fully form a TIM Barrel, about 160 are considered structurally equivalent between different proteins sharing this fold.

Alignment of sequences or structures TRNA super family. n TIM super family. n Aldolase super family n

t. RNA super family (PDBSEQ vs FASTA, Clustal. W) TIM super family (PDBSEQ vs FASTA, Clustal. W)

Aldolase super family (PDBSEQ vs FASTA, Clustal. W)

Residues n n The remaining residues that are not structural form the loop regions that connect the β strands with the α helices containing the amino acids responsible for its catalytic chemistry. The specific enzymatic activity is, in each case, determined by the loop regions at the carboxy end of the β strands (length and amino acid sequences), which do not contribute to the structural stability

Residues The number of active site residues at the eight βα motifs. The active site residues, which could be aligned using multiple structure alignment, are indicated with asterisks (*).

Super families & functions Super family Functions Triosephosphate isomerase (TIM) Isomerase Ribulose-phoshate binding barrel Isomerase / Lyase / Synthase Thiamin phosphate synthase Transferase Pyridoxine 5'-phosphate synthase Oxidoreductase FMN-linked oxidoreductases Oxidoreductase / Dehydrogenase Inosine monophosphate dehydrogenase Oxidoreductase PLP-binding barrel Isomerase / Lyase NAD(P)-linked oxidoreductase Oxidoreductase (Trans) glycosidases Hydrolase / Transferase / Glycosidase / Isomerase / Oxidoreductase Metallo-dependent hydrolases Hydrolase / Phosphotriesterase

Super families & functions Super family Functions Aldolase Lyase / Hydrolase / Transferase / Oxidoreductase Enolase C-terminal domain-like Lyase / Hydrolase / Isomerase Phosphoendpyruvate/ pyruvate domain Lyase / Kinase / Transferase / Isomerase / Synthase Malate synthase G Lyase Rub. Is. Co large subunit C-terminal domain Lyase Xylose isomerase-like Lyase / Hydrolase / Isomerase / Oxidoreductase Bacterial luciferase-like Oxidoreductase Nicotinate/Quinolinate PRTase CTerminal domain like Hydrolase / Transferase PLC-like phosphodiesterases Hydrolase Cobalamin (Vitamin B 12)dependent enzymes Lyase / Isomerase

Super families & functions Super family Functions t. RNA-guanine transglycosylase Transferase Dihydropteroate synthetase-like Transferase / Shyntase UROD/Met. E-like Transferase / Lyase FAD-linked oxidoreductase Oxidoreductase Monomethylamine methyltransferase Mtm. B Transferase Homocysteine S-methyltransferase Transferase (2 r)-phospho-3 -sulfolactate synthase Com. A Lyase Radical SAM enzymes Transferase / Oxidoreductase / Ligase Glp. P-like Transcription Cut. C-like Metal binding protein Thi. G-like Biosyntetic protein TM 1631 -like Unknown

Super families & functions (Nagano et al. 2002, Rison et al. 2000, Pujadas and Palau, 1999, Wierenga, 2001).

Active sites (general) n We have described a general relationship between structure and function for the βα barrel structures. They all have the active site at the same position with respect to their common structure in spite of having different functions as well as different amino acid sequences. n TIM barrels serve as scaffolds for active site residues in a diverse array of enzymes. Residues that form the active site are always located at the same end of the barrel, associated with the C-terminal ends of b-strands and the loops connecting these to a-helices. n In almost every one of the more than 100 different known a/b structures of open a/b sheet the active site is at the carboxy edge of the B sheet. Functional residues are provided by the loop regions that connect the carboxy end of the b strands with the amino end of the alpha helices. This is similar therefore to the a/b barrel structures.

Active sites (comparison) n The general shapes of the active sites are quite different, however. Open a/b structures cannot form funnel shaped active sites like the barrel structures. Instead, they form crevices at the edge of the B sheet. Such crevices occur when there are two adjacent connections that are on opposites sides of the B sheet. n The position of such crevices is determined by the topology of the B sheet and can be predicted from a topology diagram. The crevices occur when the strand order is reversed and can be easily identified in a topology diagram as the place where connections from the carboxy ends of two adjacent B strands go in opposite directions one to the left and one to the right.

Active sites (prediction) n Such positions in a topology diagram are called topological switch points. It was postulated in 1980 by Carl Branden, in Uppsala, Sweden that the position of active sites could be predicted from such switch points. Since then at least one part of the active site has been found in crevices defined by such switch points in almost all new a/b structures that have been determined. n Thus we can predict the approximate position of the active site and possible loop regions that form this site in a/b proteins. This is contrast to proteins of the other two main classes a helical proteins and ant parallel B proteins, where no such predictive rules have been found.

Evolution n Because the members of this family of protein catalyze a wide range of reactions and the lack of strong sequence homology, The evolutionary history of this family has been the subject of vigorous debate.

Evolution n n If members of a protein family show common function as well as a common structure, it has generally been assumed that all members have diverged from a common ancestor. If members of a protein family has no common function, It is possible that these proteins are related by convergent evolution to a stable fold.

Evolution n The evolutionary history of this family of enzymes is still not completely certain, but divergent evolution from a common ancestor explains more of the available data than does convergent evolution to a stable fold. (Reardon and Farber 1995; Petsko 2000; Gerlt and Babbitt 2001)

n Notwithstanding the diversity of their catalytic reactions, the active site is always found at the C-terminal end of the barrel sheets, suggesting divergence from an ancestral TIM barrel (Bränden & Tooze 1991). n Fifteen years ago, Farber & Petsko (1991) classified 17 TIM barrel structures into four families on the basis of their different geometries, and suggested divergence from a common ancestor. n Bränden also analyzed 19 TIM barrel structures, considering their domain organization, the metal and phosphate binding sites as well as catalytic centres. It was suggested that the presence of the common phosphate-binding site, formed by loop-7 loop-8 and a small helix (helix-8), is the strongest evidence obtained for the divergent evolutionary history of TIM barrels (Bräden 1991, Wilmanns et al 1991). n Using 30 TIM barrel structures and their sequence families, the evolution of TIM barrels was discussed by Reardon & Farber (1995), and they also agree with the divergent evolution. n More recently Copley & Bork (2000) re-analyzed a subset of the TIM barrels, including those which bind phosphate. They suggest that five of these families (two classes of aldolase; dihydropteroate synthetase; pyruvate kinase and enolase) are distantly related to the enzymes with a common phosphatebinding motif.

Evolution n n Because 3 -dimentional structure evolves more slowly than primary structure, the lack of sequence homology could be due to the age of the ancestral enzyme. The similar location of the active site in all of the members of the family, combined with geometric arguments concerning the barrel structure and the clear indications of gene duplication, followed by specialization whiting the various families, let the argued of these proteins are related by divergent evolution from a common ancestor.

The yellow markers indicate the glycosidase subgroups or family. The green color shows the enzyme families with the SPB motif, whilst the blue one shows those with phosphatemoiety of the ligands in the same position as the SPB motif. Filled markers indicate the metal-binding families, whilst open markers indicate the remainder. The triangles show seven-stranded TIM barrel family. Nagano et al 2002

Evolution of the sequence n Are new enzymes formed from random sequences generated by recombination and other genetic rearrangement or do they arise by divergent evolution from a preexisting set of enzymes. n There is debate over whether the many different TIM barrel enzymes are evolutionarily related, since in spite of the structural similarities there is tremendous diversity in catalytic functions of these enzymes and little sequence homology. n Greg Petsko provided strong evidence for the latter case from studies of a/B barrel enzymes in a rare metabolic pathway, conversion of mandelate to benzoate. This rare metabolic pathway is thought to be of recent evolutionary origin, since it’s present in only a few pseudomonad species.

Evolution of the sequence n Petsko found that the three dimensional structure of mandelate, including it’s a/b barrel, is very similar to that of a quite different enzyme, muconate lactonizing enzyme, which catalyzes a different chemical reaction, but which also involves the formation of an intermediate by proton abstraction. n The amino acid sequences of the 350 residues of these enzymes showed 26% sequence identity, which clearly demonstrates that they are evolutionary related. By comparing these two structures in detail Petsko found significant similarities in the region of the active site that catalyzes proton abstraction and intermediate formation but substantial difference in those regions of the active site that confer substrate specifity.

Evolution of the sequence n These results are compatible with an evolutionary history in which the new enzyme activity of mandelate racemase has evolved from a pre existing enzyme that catalyzes the basic chemical reaction of proton abstraction and formation of an intermediate. n Subsequent mutations have modified the substrate specifity while preserving the ability to catalyze the basic chemical reaction. Chemistry is the important factor to preserve during evolution of new enzymes, while specifity can be modified. It would therefore seem that relatively nonspecific enzymes which may have existed earlier in evolution or which may arise occasionally through random genetic rearrangements are the clay from which nature sculpts new enzymes.

Superimposition n t. RNA (PDB -> STAMP -> Ras. Mol, 2 sets) Score: 9. 87

Superimposition n Aldolase (PDB -> STAMP -> Ras. Mol, 2 sets) Score: 9. 87

Gene Fusion n A fusion gene is a hybrid gene formed from two previously separate genes. It can occur as the result of a translocation, interstitial deletion, or chromosomal inversion

Gene Fusion n Gene duplication plays an important role in enzyme evolution. It has been estimated that 50% of all genes in microorganisms are the result of duplication events, which are followed by diversification of the twin genes (Fani et al 1998, Lynch & Conery 2000).

Gene Fusion n n Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (αβ)8 -barrel, has evolved by the duplication, fusion, and mixing of (αβ)4 -halfbarrel domains (Höcker et al 2004).

Gene fusion It has been postulated that His. A and His. F evolved from a common (βα)4 -half-barrel by a series of gene duplication and diversification events (Höcker et al 2004). (βα)8 -barrels were derived from ‘half-barrels’ will motivate the search for ancestral domains within other apparent single-domain protein folds (Petsko, 2000; Gerlt and Babbitt, 2001, Jürgens et al 2000).

Birte et al. (2004)

Fold Change in Evolution of Protein Structures Mechanisms for evolutionary fold change: 1. Insertion, deletion, or substitution of structure elements 2. Circular permutation 3. Strand invasion/withdrawal 4. Beta hairpin flip/swap

Insertion, Deletion, Substitution n n Luciferase and NFP deletes a 90 -residue section that contains an αβαβα segment of TIM barrel Missing segment is compensated for by a single anti-parallel strand Shared regions are 30% identical, they are part of same operon, and homology was detected before Luciferase structure was solved Other TIM-like proteins have less dramatic changes of similar nature

Insertion, Deletion, Substitution

Circular Permutation n n Circular permutations can occur because the N- and Ctermini of proteins often end near each other As a result, elements can be substituted into structure from N- and C- termini Though structure is barely changed, topology is different, and so is different fold C 2 domains have simple permutation of only one strand Have clear sequence homology to each other, as well as very similar structures

Circular Permutation We can observe circular permutations between families of the TIM barrel super family FAD-linked oxidoreductase and also in the proteins of the super family PLPbinding barrel. n The last ones perform a circular permutation of the canonical fold. n

Circular Permutation Triosephosphate isomerase us Alanine racemase (Clustal. W)

Strand Invasion / Withdrawal n Is defined as on both sides of the invading strand disruption of Hbonds in internal βstrands, which requires changes in H-bonding patterns

Beta-Hairpin Flip / Swap • Β-hairpin flip/swap shifts location of two strands so that they have new partners on one side, and new Hbonding on the other

Implications n Two scenarios for homology in cases of a small region of similarity: Local: segments of clear homology are inserted into different structural frameworks, producing local regions of homology in the middle of nonhomologous proteins ¨ Global: shared segments of similarity are leftovers from a once completely shared structure. Gradual change of rest of structure through sequence changes and indels ¨ n All homology is local to a certain extent Structures that are mostly similar are probably mostly homologous, though alien segments could fill some sections without being detectable ¨ Structures that are very plastic and have changed considerably may only have small sections of true homology ¨ n n Homology modeling could give erroneous results in some cases Understanding the natural changes in folds could help with protein design