BMMB597EProtein Evolution

Protein classification

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Protein families

• The first protein structures determined by X-ray crystallography, myoglobin and haemoglobin, were solved (in 1959—60) before the amino acid sequences were determined

• It came as a surprise that the structures were quite similar

• Soon it became clear, on the basis of both sequences and structures, that there were families of proteins

myoglobin haemoglobin

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50 years earlier, there were some hints …

• E.T. Reichert & A.P. Brown. The differentiation and specificity of corresponding proteins and other vital substances in relation to biological classification and organic evolution: the crystallography of hemoglobins. (Carnegie Institution of Washington, 1909)

• Crystallography 3 years before discovery of X-ray diffraction?

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Reichert and Brown studied interfacial angles in haemoglobin crystals

• Stenö’s law (1669): different crystals of the same substance may have differerent sizes and shapes, but the angles between faces are constant for each substance

• They found that the angles differed from species to species

• Similarities in values of interfacial angles were consistent with classical taxonomic tree

• They even found differences between oxy- and deoxyhaemoglobin

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Most premature scientific result ever?

• These results implied:– That proteins adopted (or at least could adopt)

unique structures, to form a crystal– That protein structures varied between species– That this variation was parallel with the evolution

of the species– That proteins could change structure as a result of

changes in state of ligation• In 1909!

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M.O. Dayhoff

• Pioneer of bioinformatics• Collected protein sequences• First curated ‘database’• Recognized that proteins form families, on the

basis of amino acid sequences• Computational sequence alignments• First evolutionary tree • First amino-acid substitution matrix (later

replaced by BLOSUM)

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Can relationships among proteins be extended beyond families?

• Families = sets of proteins with such obvious similarities that we assume that they are related

• One question: how much similarity do we need to believe in a relationship?

• How far can evolution go?• Convergent evolution?• Cautionary tale: chymotrypsin / subtilisin

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Chymotrypsin-subtilisin

• Both proteolytic enzymes– Chymotrypsin mammalian– subtilisin from B. subtilis

• Both have catalytic triads• Same function – same mechanism• Sequences 12% similar (near noise level)

• However, structures show them to be unrelated

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Chymotrypsin / Subtilisin

Catalytic triad in serine proteinases

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Chymotrypsin and subtilisin have similar catalytic triads

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How can we classify proteins that belong to families?

• Align sequences• Calculate phylogenetic tree (various ways to

do this, depend on sequence alignment)• Usually, phylogenetic tree of homologous

proteins from different species follow phylogenetic tree based on classical taxonomy

• That is reassuring• But what happens as divergence proceeds?

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How can we classify proteins that do not obviously belong to families?

• Base this on structure rather than sequence• Structural similarities are maintained as

divergence proceeds, better than sequence similarities

• For closely related proteins, expect no difference between sequence-based and structure based classification

• How far can classification be extended?

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SCOP Structural Classification of Proteins

• Idea of A.G. Murzin, based on old work by C. Chothia and M. Levitt

• Even if two proteins are not obviously homologous, they may share structural features, to a greater or lesser degree.

• For instance, the secondary structures of some proteins are only -helices

• Others, have -sheets but no -helices

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SCOP

• SCOP is a database that gives a hierarchical classification of all protein domains

• Recall that a domain is a compact subunit of a protein structure that ‘looks as if’ it would have independent stability

Fragment of fibronectin

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Dissection of structure into domains

• It is not always quite so obvious how to divide a protein into domains

• There is some (not a lot) of room for argument• Note that sometimes the chain passes back

and forth between domains• In these cases one or both domains do not

consist entirely of a consecutive set of residues

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lactoferrin

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SCOP, CATH, DALI Database classify protein structures

• SCOP (Structural Classification of Proteins) • CATH (Class, Architecture, Topology, Homologous

superfamily)• DALI Database • These web sites have many useful features: – information-retrieval engines, including

search by keyword or sequence– presentation of structure pictures– links to other related sites including bibliographical

databases.

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SCOPhttp://www.scop.mrc-lmb.cam.ac.uk

• SCOP organizes protein structures in a hierarchy according to evolutionary origin and structural similarity.

• Domains -- extracted from the Protein Data Bank entries.

• Sets of domains are grouped into families: sets domains for which imilarities in structure, function and sequence imply a common evolutionary origin.

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The SCOP hierarchy

• Families that share a common structure, or even a common structure and a common function, but lack adequate sequence similarity – so that the evidence for evolutionary relationship is suggestive but not compelling – are grouped into superfamilies

• Superfamilies that share a common folding topology, for at least a large central portion of the structure, are grouped as folds.

• Finally, each fold group falls into one of the general classes.

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Major classes in SCOP

• – secondary structure all helical• – secondary structure all sheet• / – helices and sheets, but in different parts of

structure• + – contain -- supersecondary structure• ‘small proteins’ – which often have little

secondary structure and are held together by disulphide bridges or ligands; for instance, wheat-germ agglutinin)

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Summary of SCOP hierarchy

• Class• Fold• Superfamily• Family• Domain

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SCOP classification of flavodoxin

Protein: Flavodoxin from Clostridium beijerinckii [TaxId: 1520]Lineage:Root: scop Class: Alpha and beta proteins (a/b) [51349] Mainly parallel beta sheets (beta-alpha-beta units) Fold: Flavodoxin-like [52171] 3 layers, a/b/a; parallel beta-sheet of 5 strand, order 21345 Superfamily: Flavoproteins [52218] Family: Flavodoxin-related [52219] binds FMN Protein: Flavodoxin [52220] Species: Clostridium beijerinckii [TaxId: 1520] [52226] PDB Entry Domains:5nul complexed with fmn; mutant chain a [31191]

2fax complexed with fmn; mutant chain a [31194]

… many others

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Clostridium beijerinckii Flavodoxin(stereo pair)

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Flavodoxin NADPH-cytochrome P450 reductase

same superfamily, different family

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Flavodoxin CHEY same fold, different superfamily

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Flavodoxin Spinach ferredoxin reductase

same class, different folds

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Flavodoxin in the SCOP hierarchy• To give some idea of the nature of the similarities expressed by the

differentlevels of the hierarchy

• Flavodoxin from Clostridium beijerinckii and NADPH-cytochrome P450 reductase are in the same superfamily, but different families.

• Flavodoxin and the signal transduction protein CHEY are in the same fold category, but different superfamilies.

• Flavodoxin and Spinach ferredoxin reductase are in the same class – + – but have different folds.

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CATH presents a classification scheme similar to that of SCOP

• CATH = Class, Architecture, Topology, Homologous superfamily, the levels of its hierarchy.

• In CATH, proteins with very similar structures, sequences and functions are grouped into sequence families.

• A homologous superfamily contains proteins for which similarity of sequence and structure gives evidence of common ancestry

• A topology or fold family comprises sets of homologous superfamilies that share the spatial arrangement and connectivity of helices and strands

• Architectures are groups of proteins with similar arrangements of helices and sheets, but with different connectivity. For instance, different four -helix bundles with different connectivities would share the same architecture but not the same topology in CATH

• General classes of architectures in CATH are: . , - (subsuming the / and + classes of SCOP), and domains of low secondary structure content.

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Do different classification schemes agree?• To classify protein structures (or any other set of objects) you

need to be able to measure the similarities among them. • The measure of similarity induces a tree-like representation of

the relationships. • CATH, SCOP, DALI and the others, agree, for the most part, on

what is similar, and the tree structures of their classifications are therefore also similar.

• However, even an objective measure of similarity does not specify how to define the different levels of the hierarchy.

• These are interpretative decisions, and any apparent differences in the names and distinctions between the levels disguise the underlying general agreement about what is similar and what is different.