Fold Change in Evolution of Protein Structures

Nick V. Grishin

Structural Drift: a Possible Path to Protein Fold Change

S. Sri Krishna and Nick V. Grishin

Protein SuperfamiliesProtein Superfamilies•Protein superfamilies: have a shared genetic ancestry (true homology)

•Evolution is economical: once a scaffold is found it is re-used over and over

•Structure is generally much more conserved than sequence, robust to sequence change

•Because of changed function/random mutation, sequences drift apart

•Structural implications of changes in sequence in a superfamily:

•Traditional assumed results:

•Protein folds in a similar way, either with stable fold, or reduced stability

•Protein misfolds

•Third possibility:

•Protein folds differently

Paths to structure changePaths to structure change•One pathway demonstrated experimentally by Cordes et al. in 1999

Arc

“Switch Arc”

Paths to structure changePaths to structure change• Grishin 2001, definition of fold: “proteins are classified within the same fold if they have the

same major secondary structural elements in the same mutual orientation and with the same connectivity (topological connections)”

• Proteins of same fold are not necessarily homologous: convergence is possible

• Conversely, proteins of different folds are not necessarily non-homologous in all cases

• If fold is different, how do we know that proteins are homologous?:

• Significant homology detected in profile searches

• Shared functional sites

• Co-occurrence of multi-domain architectures, with structural changes in one of the domains

• Grishin’s four 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, SubstitutionInsertion, Deletion, Substitution•Luciferase and NFP

•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, SubstitutionInsertion, Deletion, Substitution•Luciferase/NFP is an extreme example, most indels are short and occur at structure periphery

•Small changes in structure can occur, provided they complement core and link sections of protein together

•Sauer’s group proved that strand to helix is possible

Insertion, Deletion, SubstitutionInsertion, Deletion, Substitution•Lactate Dehydrogenase and NADH Peroxidase

•PSI-BLAST can easily detect homology, up to 26% identity between some structures, shared substrate binding region

•Two interesting structure changes:

•Helix C in LDH converts to β-meander in NADH peroxidase

•NADH has a second domain that causes strand e and helices D and E to be swapped

Insertion, Deletion, SubstitutionInsertion, Deletion, Substitution•Rossmann fold-like domains of ATP-grasp proteins

•Sequence similarities in ATP-binding site (not shown) demonstrate homology

•Separate N-terminal domain (shown) binds substrate and has a variety of structural changes

•4 proteins (b-e) can be linked to each other through small structure replacements

•Carboxypeptidase A (a) is probably also related to these domains, and has additional elaborations

b c d ea

Insertion, Deletion, SubstitutionInsertion, Deletion, Substitution•A possible path can be posited for conversion from all-β to all-α, solely through small changes seen in previous examples

•However, some steps cannot be demonstrated to have actually happened (black arrows)

Circular PermutationCircular Permutation•Circular permutations can occur because the N- and C-termini 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

•C2 domains have simple permutation of only one strand

•Have clear sequence homology to each other, as well as very similar structures

Circular PermutationCircular Permutation•Saposins have a much more striking permutation seen in the structure of aspartic proteinase prophytepsin: called “swaposin”

•Half of chain is involved in permutation, but it was detectable by sequence before swaposin structure became available

•Coherent explanation has been offered for swaposin:

•Saposins exist in tandem repeats fused by linkers

•Swaposin is probably a fusion of the C-term of one repeat, the linker, and the N-term of the next

•This tandem repeat scenario seems to be a common source of permutations

•Grishin: “Circular permutation may offer a mechanism for generating diversity analogous to recombination”

LinkerSaposin Saposin

Swaposin

Strand Invasion/WithdrawalStrand Invasion/Withdrawal•Strand invasion: defined as disruption of H-bonds in internal β-strands, which requires changes in H-bonding patterns on both sides of the invading strand

•Lipocalins display a hairpin invasion in some structures

•Result is slightly larger barrel that can bind larger lipid molecule

Strand Invasion/WithdrawalStrand Invasion/Withdrawal•Serpins (serine protease inhibitors) display strand invasion not through evolution, but in the same molecule as a structural shift

•When bound by protease target, serpin conformation shifts to hold protease in inactive state

•Protein is “metastable” having two stable conformations

•This metastable state could be used by some proteins as the intermediate evolutionary state between one fixed structure and another

•Small mutations would upset balance and fix one state permanently

Strand Invasion/WithdrawalStrand Invasion/Withdrawal•Structures of P-loop ATPases appear to be the result of a metastable state in an intermediate structure

•Location of strand a in the central sheet is changed between RecA and Adenylate Kinase

•Result is distinctive structure, even though sequence similarity is easily detectable

•Also have a small circular permutation at helix E

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

•Lipocalins are usually all anti-parallel, but thrombin inhibitor has a strand swap that adds a crossing loop, and makes two of the interactions in the sheet parallel

•Change has functional impact, as it creates a loop that blocks the entrance to the barrel (binding pocket for other lipocalins)

•Thrombin inhibitor instead acts as protease inhibitor

ImplicationsImplications•Changes within domains mean that they are not the only units of homology, homology can exist at the subdomain level

•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 non-homologous 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

•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

•Homology modeling could give erroneous results in some cases

•Understanding the natural changes in folds could help with protein design

ImplicationsImplications•Structure classification becomes more challenging:

•Phenetic classification: looks only at structure (more like CATH)

•Phyletic classification: looks at evolutionary relationships (more like SCOP)

•Phyletic classification is more useful, but more easily confounded

•SCOP is organized incorrectly:

•Currently:

•Class (overall structure type)

•Fold (overall fold similar, but not necessarily homologous)

•Superfamily (distant homology)

•Family

•Should be:

•(?) Class (overall structure type)

•Superfamily (distant homology)

•Fold (overall fold similar)

•Family

Structural DriftStructural Drift•Probably a subset of indel/substitution scenario

•Seen in formate dehydrogenase (FDH) small subunit

•Two ferredoxin domains are present, one inserted within the other

•First domain has additional structure that resembles a β-grasp fold (superimposes with low RMSD, but almost certainly analogous)

•Additional structure pulls strands a and d away from their normal position, as compared to the second ferredoxin domain

•Essentially creates a new core

•Deletions could yield a β-grasp fold from this core, hence a new fold by structural drift

•(a) Small subunit of FDH, first ferredoxin domain

•(b) Duplicated ferredoxin domain inserted within first

•(c) Same as (a), but with β-Grasp like portion colored

•(d) β-Grasp fold of protein L