Báo cáo Y học: The evolution of monomeric and oligomeric bc-type crystallins potx

R E V I E W A R T I C L EThe evolution of monomeric and oligomeric bc-type crystallins Facts and hypotheses Giuseppe D’Alessio Dipartimento di Chimica Biologica, Universita` di Napoli Fe

R E V I E W A R T I C L E

The evolution of monomeric and oligomeric bc-type crystallins

Facts and hypotheses

Giuseppe D’Alessio

Dipartimento di Chimica Biologica, Universita` di Napoli Federico II, Naples, Italy

The case of homologous monomeric c-type and oligomeric

b-type crystallins has been described and analyzed in

evo-lutionary terms.Data and hypotheses from molecular

gen-etics and structural investigations converge and suggest a

novel three-phase model for the evolutionary history of

crystallin-type proteins.In the divergent cascades of

mono-meric and oligomono-meric crystallins, a pivotal role was played by

alterations in the gene segments encoding the C-terminal

extensions and the intermotif or interdomain linker peptides These were genomic hot spots where evolution experimented

to produce the modern variety of bc-crystallin-type quater-nary structures

Keywords: crystallins; evolution; quaternary structure; introns late; introns early

The question of how oligomeric proteins evolved has gained

renewed interest in the last few years [1–9].Although the

possibility cannot be excluded that some proteins emerged

first as functional aggregates and later dissociated into

functional monomers, the available evidence suggests that

divergent evolution more often used the association of

protein protomers into oligomers to vary and enrich the cell

repertoire of structures and functions.Evidence for this

evolutionary path can be seen in the Ôhydrophilic effectÕ

recorded at intersubunit interfaces [3], i.e a surprising,

significant presence of polar and charged residues at

oligomeric interfaces.This can be readily interpreted as

the result of the association of previously exposed,

hydro-philic surfaces (from a monomer) into solvent-excluded

interfaces (in an oligomer)

It has been argued that the alteration of a protein surface

to render it adhesive for the generation of oligomers, would

be too long an evolutionary process, as it would require

multiple mutations in the gene encoding the ancestral

monomer [9].It was therefore proposed that evolution used

pre-existing interdomain interfaces that after a ÔswapÕ of

domains between monomers would be readily reconstituted

as intersubunit interfaces.This would induce the association

of monomers into oligomers without the need for a lengthy

process of substituting one residue after another to build an

adhesive interface.However, it has been noted that a swap

of domains between monomeric ancestors is not an

evolutionary event per se, but rather the outcome of one

or more mutational events in the monomeric ancestor: these

events could then prime a swap of domains [3].Monomeric

proteins have been transformed artificially into dimers by inducing the displacement of terminal helices, which deter-mined the helix segments between two monomers [10–12] However, to make the swap permanent and the dimers stable, mutations had to be engineered into the cDNAs encoding the proteins [11,12]

These were, naturally, experiments of in vitro evolution, in which a single genetic alteration was sufficient to induce oligomerization.When we compare a present-day set of homologous proteins, one monomeric the other oligomeric, what we see when we compare the amino-acid sequences of the two proteins are merely amino-acid substitutions.Some

of these may not related at all to the monomer to oligomer transition, and it is difficult and risky to discern the changes presumed to be significant for the transition.However, if we could have observed the entire process of evolution of a monomeric protein into a dimer, we would have assigned to each gene alteration responsible for the evolutionary transition a different status in the evolutionary mechanism

A ÔprimaryÕ mutation would top the hierarchy, as the single event responsible for the step of no return towards the new, oligomeric structural organization.Although such a primary event would have been essential, it may not have been sufficient to engender oligomerization.On the other hand, it may not be easy, or even possible, to decipher in the structure of a present-day oligomer what was the primary mutation originally responsible for oligomerization Besides investigations of mutational events as revealed by amino-acid substitutions in homologous proteins, another tool might be useful to shed light on putative ancestors of present-day protein oligomers.It has been surmised [3,8] that the analysis of the refolding mechanism by which denatured, unfolded polypeptide chains fold back into oligomers may shed light on the evolutionary history of the oligomers, as this might be recapitulated in the pathway of oligomer refolding

The monomeric c-crystallins and the evolutionarily related dimeric b-crystallins provide an interesting case study in the discussion of the evolutionary transition from monomeric to oligomeric proteins.They are one of the

Correspondence to G.D’Alessio, Dipartimento di Chimica Biologica,

Via Mezzocannone, 16, 80134 Napoli, Italy.

Fax: + 39 081 5521217, Tel.: + 39 081 2534731,

E-mail: dalessio@unina.it

Abbreviations: EDSP, epidermis differentiation-specific protein; TKR,

tyrosine kinase receptor.

(Received 17 December 2001, revised 8 April 2002,

accepted 17 May 2002)

present-day sets of monomeric and dimeric homologous

proteins on which the Ô3D domain swapping modelÕ has

been based [9], and special attention has been given to their

evolutionary history [13–15]

Crystallins were so named when they were recognized as

the proteins that provide the crystalline lens of the vertebrate

eye with its indispensable transparency and unique

refract-ive properties (reviewed in [16]).They are long-lrefract-ived

proteins, as lens cells live as long as their host organisms

They also have lived a very long evolutionary history, as

their primitive ancestors can be traced back to the

diver-gence of protozoa.Crystallins are not confined to the lens;

in various taxa, crystallin genetic material was ÔrecruitedÕ in

different tissues to encode proteins serving functions, as

diverse as those of enzymes and antistress proteins [17–19]

Three major classes of crystallins are common to the eye

lens throughout the vertebrates: the a-, b- and c-crystallins

The latter two classes are made up of homologous proteins,

and constitute the superfamily of bc-crystallins.For

c-crystallins, six genes (cA to cF) have been identified,

encoding 21-kDa monomeric proteins, and a gene for a cS

crystallin, previously classified as bS-crystallin b-crystallins,

encoded by five to seven genes, depending on species, may

form aggregates of up to 200 kDa that consist of acidic type

(bA1 to bA4) and basic type (bB1 to bB3) subunits,

23–33 kDa.The c-crystallins have short C-terminal peptide

extensions, whereas b-crystallins possess long N-terminal

extensions, and the basic b-type subunits also have

C-terminal extensions

In this review, only genetic and structural aspects of

monomeric or oligomeric bc-crystallins likely related to

their evolutionary origin will be discussed.For other aspects, the reviews cited above should be consulted

S T R U C T U R A L F E A T U R E S O F

M O N O M E R I C A N D D I M E R I C

C R Y S T A L L I N S

To date, the available 3D structures are those of bB2-crystallin [20–22], and of cB- [23–25], cE- [26], and cC-crystallin [27].Formerly, the latter were called cII-, cIIIB- and cIV-crystallin, respectively cB-crystallin is monomeric, as are all c-crystallins; bB2-crystallin is a dimer

in solution, although its structural unit in the crystal lattice is

a tetramer, made up of two dimers, and the likely assembly

of this protein in the lens is that of higher heteroligomers However, monomeric cB-crystallin and dimeric bB2-crys-tallin will be considered here as the monomeric and dimeric prototypes for the respective families of c- and b-crystallins, and simply referred to as c-type or b-type crystallins, respectively

Both monomeric c-type crystallin and the subunit of dimeric b-type crystallin are composed of two domains, an N- and a C-terminal domain (termed N- and C-domains) Each domain is made up of two homologous ÔGreek keyÕ

b strand motifs; motifs M1 and M2 in the N-domain, and motifs M3 and M4 in the C-domain.In both the c-monomer and the b subunit, the four motifs and the two domains are organized symmetrically, with local intermotif and interdomain pseudo-dyads.However, in dimeric b-crystallin the topological equivalents of the two domains of monomeric c-crystallin are domains from

Fig 1 The structures of: (A) bB2-crystallin

(PDB 1BLB),the dimeric b–type prototype,

and (B) cB-crystallin (PDB 4GRC) The

monomeric c-type prototype.Fragments of

cB-crystallin are shown to illustrate

schemat-ically the structures of (C)

two-motif/one-domain, and (D) one-motif putative crystallin

ancestors.The interdomain linker peptides are

colored in red.

different subunits (see Fig.1).Two types of dimers may

thus be viewed in a b-type crystallin: the orthodox dimer,

made up of the two subunits with the two N• C and N¢ • C¢

domains covalently linked through linker peptides (|) in

antiparallel fashion:

and a pseudo-dimer made up of noncovalently associated

N• C¢ and C • N¢ domains from the two subunits, which

reproduce the topological association of the N- and

C-domains of c-crystallin (see Fig.1).The linker peptide

segments that connect the N- and C-domains have very

different conformation in the c-monomer and in the

b subunits.In the monomeric c-type crystallins, the linker

peptide bends to reach from the N-domain through the

C-domain as in N• C.In dimeric b-type crystallin instead,

the two linker peptides have an extended conformation; as

in the scheme above, they run antiparallel on either side of

the pseudo twofold axis relating the two-domain

pseudo-dimeric structure made up of N• C¢ and C • N¢ (see Fig.1)

When present, N- and C-terminal extensions are not

entirely defined in the structure of crystallin proteins, as they

are flexible, without unique conformations, with the

excep-tion of the proximal segments of the C-terminal extensions

M O L E C U L A R G E N E T I C S S T U D I E S :

F A C T S A N D H Y P O T H E S E S

Owing to the stringent necessity to conserve the critical

function of providing the lens, by an appropriate

arrange-ment of protein aggregates, with the precision of an optical

measuring instrument, lens crystallins have been subjected

to severe selective pressure in the course of their evolution

This is indicated by the very low substitution rates registered

in the vertebrate crystallin genes, especially in those coding

for b-crystallins, and by the unusually very similar

substi-tution rates recorded for internal and surface regions of

these proteins [14].The latter finding can be interpreted as

indicative of the importance of surface, intermolecular

interactions among the lens proteins

A striking exception to this general sequence conservation

rule are the high substitution rates that have been recorded

only for the sequences encoding the interdomain linker

peptides and the N- and C-terminal extensions.These

findings certainly have an evolutionary significance

In both b- and c-type crystallin genes, the sequence

coding for the interdomain linker peptide is interrupted by

an ÔinterdomainÕ intron.In the b-type crystallin genes,

ÔintermotifÕ introns are also present.Thus in b-type genes,

each motif (M1, M2, M3, M4) is encoded by a separate

exon, whereas in the c-type genes the pairs of adjacent

motifs (M1/M2 and M3/M4) are each encoded by a single

exon (Fig.2).Sequence similarities are higher when motif

M1 is compared with M3 (termed A type motifs), or M2

with M4 (B type motifs), which results in a ABAB pattern

[35]

The structural similarity and topological equivalence

between motifs and between domains, and the significant

degree of sequence identity between domains, even higher

than between motifs, led to the proposal [23] that the evolutionary path of c-crystallin started with a one-motif ancestor.Then, upon gene duplication followed by fusion, a two-motif/one-domain protein evolved, to be followed, after a second duplication-fusion step, by the two-domain c-type proteins.Subsequent findings from protein sequence, gene sequence, and structural studies [7,13,14,20] have strengthened and expanded this view

This evolutionary path based on primary and tertiary structure homologies, and consisting of two main events of gene duplication, each followed by fusion, is supported by the identification in distant phyla of homologous genes encoding proteins that can be related to putative crystallin ancestors.A one-domain crystallin-like fold has been found

in a protein (spherulin 3a) from a slime mould, with a significant sequence identity and a high structural similarity with c-crystallin domains [61,65].Interestingly, in the amino-acid sequence of spherulin 3a motif M1 is not N-terminal as in bc-crystallin sequences, but C-terminal to motif M2 (Fig.2).Another case of a one-domain crystallin fold has been identified [30] in Streptomyces metallo-proteinase inhibitor (SMPI), with a clear relationship in three-dimensional structure to bc-crystallins.In this protein,

a significant, albeit weak, sequence similarity has been detected between its N-terminal motif and M1 motif of bc-crystallins, but no similarities were found between its

Fig 2 A scheme of the arrangements of motif encoding gene sequences

in crystalline-type genes SPHE-, STRE-, S-, C-, b- and c-type nota-tions indicate motif arrangements in: spherulin 3a, Streptomyces protease inhibitor, S-protein-, G Cydonium protein, b- and c-type crystallin, respectively.Motifs are shown as boxes and their numbers (M1 through M4) are those typical of both b-type and c-type crys-tallins, assigned to the other genes on the basis of homologies.Two-motif domains are formed by adjacent homologies.Two-motifs.Thin and thick bars represent intermotif and interdomain introns, respectively.Dotted line segments between domains or motifs indicate that it is not known if an intermotif or an interdomain intron is present in that gene.

C-terminal domain and any other known crystallin-type

motif sequences (in Fig.2, this motif is marked as MX).A

crystallin-type one-domain fold has also been proposed for

a yeast toxin [31], and for a Streptomyces toxin-like protein

[32].However, in these cases the possibility of convergent

evolution may not be excluded [33]

Two-domain crystallin-like folds have also been found

One was identified in protein S from the spore coat of a

bacterium [34], another long-lived protein (like spherulin 3a

and the crystallins).Interestingly, in this two-domain

protein, the four homologous motifs are not arranged as

in bc-crystallins (M1-M2-M3-M4), but in a reversed pattern

(M2-M1-M4-M3) (Fig.2).This prompted the suggestion

[13] that the two evolutionary lines of the bacterial

crystallin-like protein and the vertebrate crystallin ancestor

diverged at the one-motif stage

Another two-domain, evolutionarily related member of

the bc-crystallin superfamily has been identified in the

epidermis differentiation-specific protein (EDSP) from an

amphibian, Cynops pyrrhogaster [35].The N-terminal

portion of this protein contains four crystallin-type motifs

that appear to be arranged in the M1-M2-M3-M-4 pattern

typical of the b- and c-type lens crystallins.More recently, a

two-domain crystallin signature has been identified in a

protein sequence from a sponge of the genus Geodia [36].In

this protein too the four Greek key motifs are arranged

in the same order (M1-M2-M3-M4) as in the vertebrate

bc-crystallin genes

Another impressive addition to the bc-crystallin

super-family is that proposed for AIM1, a protein encoded in a

human gene whose expression has been related to

melan-oma suppression [37].The 3¢ terminal region of this gene

codes for a protein sequence comprising 12 crystallin-type

motifs arranged in the M1-M2-M3-M4 order.Trimeric

protein models have been constructed connecting the six

two-motif domains with either the bent c-type or the

extended b-type interdomain linkers.The gene, however,

appears to code for protein domains more closely related to

b-type than to c-type crystallins.This conclusion is based on

the following elements: (a) the linker peptide sequences are

closer to those typical of b-type crystallins; (b) the gene

contains intermotif introns as the b-type genes; (c) the

interdomain intron positions are homologous to those of

the b-type crystallins introns

As for the evolution of dimeric b-type crystallins, the

possibility that a c-type gene encoding a monomeric

crystallin was the immediate ancestor to a b-type gene

encoding a dimeric crystallin has been excluded [14], based

on the absence of intermotif introns in c-crystallin genes and

their presence in b-type genes (Fig.2).The lack of these

introns in c-crystallins has been attributed to an intron loss

occurred in a two-motif/one-domain crystallin ancestor

The loss would have occurred in the c-type genes only after

the divergence of the evolutionary paths leading to c-type

and b-type genes, respectively.This because it was deemed

unlikely that an identical mutational event, the intron loss,

could have occurred twice in the evolution of two

homol-ogous one-domain genes after their divergence and before

their fusion into four-motif/two-domain encoding genes

In fact, the opposite argument may be valid.The

probability that a certain type of gene alteration occurs

(an insertion, a deletion) depends on extrinsic (e.g nature of

the mutagen, environmental conditions) and on intrinsic

factors: the base sequence, the consequent secondary and supersecondary structures, as well as the topology of the DNA region in which the event takes place.For homolog-ous genes we may assume that they share most of the intrinsic and extrinsic elements.Thus to evaluate the probability that a certain gene alteration occurred in evolution in homologous genes, we should use very similar probability factors.In conclusion, the likelihood that a gene alteration, such as an intron loss or insertion, could occur twice in the evolution of homologous genes in a certain gene family is greater the closer these genes are in evolution, i.e if their divergence was a recent occurrence

Furthermore, the proposal that an intermotif intron loss occurred at the two-motif/one-domain stage would have as

a consequence that a single two-motif/one-domain-enco-ding gene, in which the loss would have occurred, was the common ancestor to all subsequently diverged one-domain genes.But this does not appear to be the case, as indicated

by the different motif orders in different crystalline-type genes.As mentioned above, in cb-type crystallins homol-ogous M1 motifs are N-terminal to motif M2, whereas in spherulin 3a and in protein S they are C-terminal to M-2 (see Fig.2).Hence, in the evolutionary path of crystallin-type proteins it would seem unlikely that a single two-motif/ one-domain ancestor duplicated and diverged while also undergoing a switch of motif-encoding sequences to gener-ate different motif arrangements in the various descendant genes

A more general argument in favour of a late insertion of introns in crystallin-type genes, as opposed to a late deletion

of pre-existing introns, may be based on the intron-late theory, originally proposed to explain the presence of spliceosomal introns in eukaryotes, and their absence in archea and in eubacteria [38–40].Over recent years, a vast amount of data has been interpreted as supporting this theory [41].In particular, the results of a statistical analysis [42] of pairs of gene paralogs may only be interpreted to favour intron gains rather than intron losses in these genes Recent data [43] in support of the theory is the finding that

in the sponge Geodia the gene encoding the extracellular and transmembrane domains of the tyrosine kinase receptor (TKR) has no introns.In homologous vertebrate TKR genes instead several introns are present

As for the late insertion of introns in crystallin-type proteins, it has been recently found (A.Di Maro, M.V Cubellis & G.D’Alessio, unpublished results) that there are

no introns in the gene encoding the crystallin-type protein from Geodia (see above).It should be noted that Geodia sponges are very primitive organisms that diverged more than 500 million years ago (some 300 million years earlier than mammals), whose crystallin genes have a full comple-ment of introns.This finding is in support of late gains of introns, rather than introns loss in the evolution of crystallin-type genes

An alternative model may therefore be proposed for the evolution of crystallin-type genes, clearly evolved from the previous models reported above [13,14,23].In this model an early one-motif crystallin ancestor gene duplicated and diverged into several one-motif genes, whose combinatorial fusion engendered several two-motif pairs.This would accommodate all the motif arrangements (M1-M2, M1-M3, etc.) identified in present time crystallin-type proteins (Fig.3).In this scenario there would be no loss of intermotif

introns in the evolutionary pathway to c-type genes, but

rather the acquisition of an interdomain intron in the

evolution of c-crystallins, and of both interdomain and

intermotif introns in the evolution of b-type crystallin genes

(Fig.3).The likelihood of late-in-evolution intron insertion,

or that identical mutational events could have occurred in

evolutionarily proximal genes has been discussed above

It should be noted that the DNA sequences coding for

C-terminal and N-terminal extensions in one-domain

ancestral crystallin genes would be the most likely

candi-dates for the formation of intron sequences between motifs

or between domains.This can be based not only on their

intermotif and interdomain topologies, but also on their

very high substitution rates (see above)

Thus the available evidence from molecular genetics

studies on crystallin-type genes may be interpreted as

illustrated in Fig.3, in which three main phases are

summarized.In phase 1, duplications and divergence of

the putative earliest, one-motif M ancestor occurred.In

phase 2, the diverged duplicates fused in different

combi-nations and underwent further divergence.Upon fusion, an

intron formed between motifs in the evolutionary path of

the ancestors toward the b-type, but not in that toward the

c-type genes.In phase 3, the two-motif/one-domain

enco-ding genes duplicated and fused, with the formation of an

interdomain intron, possibly after the divergence of

verte-brates

It should be noted that the scheme illustrated in Fig.3

provides parallel, independent evolutionary paths for c-type

monomers and for b-type oligomers.Thus, as previously

proposed [14], oligomeric b-type crystallins did not evolve

from monomeric c-type crystallins, although here this

conclusion is based on different considerations.Naturally,

and in line with previous analyses [2,3], such conclusion

excludes the possibility that a dimeric b-type crystallin

evolved from a monomeric c-type crystallin through a 3D

domain swap [9]

The molecular genetics studies described above also

suggest an important evolutionary role of the DNA regions

encoding the interdomain linker peptides and the terminal extensions, as they are regions: (a) with high substitution rates; (b) where intron insertions or deletions occurred

S T R U C T U R A L S T U D I E S : F A C T S

A N D H Y P O T H E S E S When the question of crystallin evolution is examined from

a structural viewpoint, the most impressive data is the high conservation of hydrophobic patches at inter–domain interfaces [20,23].In c-crystallin, the hydrophobic residues Met43, Phe56 and Ile81 from motif M2 interact with the homologous Val132, Leu145 and Val170 from motif M4 Identical or analogous interactions occur at the b-crystallin interface between the triad of Val55, Val68, and Ile92, and that of Val143, Leu156, and Ile181.Then the C-terminal extensions have also been suspected to have a role in the evolution of domain association, as suggested by the interdomain hydrophobic interactions observed between the C-terminal extensions of the b-C-domain and the surface of the N-domain from the partner subunit [20], and

by the peculiar behaviour [44] of the isolated c-C-domain altered at its C-terminal extension (see below).Finally, the strikingly different conformations of the interdomain linkers, bent or extended in c- and b-crystallins, respectively, could certainly not escape attention

Thus, the key structural features to focus upon while analyzing the evolution of two-domain or two-subunit bc-crystallins (i.e the determinants of interdomain association, intramolecular or intermolecular), are the hydrophobic interdomain patches, the interdomain linker peptides, and the terminal extensions.These have been the precise targets selected by the London and Regensburg research groups in their investigations on the structural determinants and the evolution of the b-type and c-type crystallins [7,25,44–51] The burying of the hydrophobic patches at the interdo-main interfaces, intramolecular in the c-type structure, intermolecular in the b-type, has been early recognized as the apparent driving force for domain association [20,23]

Fig 3 A schematic summary of the main events in the evolutionary paths leading to pre-sent-day crystallin-type genes M denotes a monomeric putative ancestor encoding a one-motif (Greek key) protein, hyphenated to indicate duplication and divergence of genes, with the numerals 1–4 indicating motif typologies.Subscripts F, S, C, G, and B denote the respective evolutionarily com-mitted ancestors of: spherulin 3a, protein S, the G cydonium protein, the c-type, and the b-type crystallin genes.The segments con-necting the boxed M motifs indicate the pres-ence of intermotif (thin bars) and interdomain (thick bars) introns; the lack of separation lines between motifs or domains indicate that

in those cases the presence or absence of introns has not been determined.

However, an impressive network of H-bonds and ion pairs

between Glu and Arg residues is also evident in these

structures at the interdomain interfaces [52].It is therefore

tempting to conclude that the polar or charged side-chains

involved in these contacts are remnants of the ancestral,

solvent exposed surfaces of single-domain crystallins, now

buried at interdomain interfaces of present day crystallins

As they concur to the interface stabilization, we can suggest

that a Ôhydrophilic effectÕ [3] apparently concurred in

stabilizing the interfaces of crystallins that evolved into

higher order structures

As for the hydrophobic patches, many experiments have

been performed to investigate their importance in the

determinism of domain association, some of them with

contradicting results.It has been reported that isolated

c-crystallin domains, perfectly equipped with their

hydro-phobic triad, either obtained through proteolytic cleavage

[53], or as recombinant proteins [54], do not associate

spontaneously into c-like domain dimers, and behave as

stable monomeric proteins.These results would lead to

conclude that the hydrophobic effect is not the only

determinant of domain association.Yet, they may simply

suggest that covalent interdomain linkers are essential to

raise the local concentration of interdomain surfaces and

engender the hydrophobic effect [46].On the other hand, the

substitution of a single residue (Phe56, replaced by Ala, Asp

or Trp) in the triad responsible for the hydrophobic patch

proved sufficient to destabilize c-crystallin domains to the

point of rendering them incapable of engaging into a stable

association [48]

Different results have been obtained with the isolated

N-domain of rat bB2-crystallin, found to associate in

solution [51], and with the isolated N- and C-domains of

c-S-crystallin, for which a tendency to associate into

heterodimers has been reported [55].It should be noted

that c-S-crystallin is very similar to b-crystallin, and that for

a long time it was labelled as a b-crystallin.Recently, the

structure of dimeric N-domains from rat bB2-crystallin has

been solved [52] and shown to be maintained essentially by

the canonic hydrophobic contacts described above, and by

the polar interactions mentioned above

The apparent discrepancy between the two sets of data

may be reconciled by the conclusion that c-type domains,

once dissociated cannot re-associate, whereas domains of

b-type and b-like c-S-type crystallins do not need a high

local concentration of structural elements to build up the

interface.Hence, in c-type crystallins the interdomain

hydrophobic patches may not be the only determinant for

domain association, whereas they are determinant and

sufficient in b-crystallins.This conclusion may not be

surprising if we consider the radically different

conforma-tion of the interdomain linker peptides, bent and extended,

respectively, in c-type and b-type crystallins.In the

former case, the bent linker seems to be essential to drive

the association at the interface, whereas in the latter the

extended, spatially distant linker is not involved in the

association

The role of the linker peptides in the determination of

monomeric vs.dimeric structures, has also been investigated

by protein engineering, with apparently contradicting

results.One early conclusion had been that the linker

peptides have no role in determining domain association

This was based on the following findings: a c-type protein

remains monomeric when its c-type linker is replaced by a b-type linker [46].Likewise, a b-type protein remains a dimer when its original linker is replaced with a c-type linker [56], as described previously [49].In these experiments, the exchanged sequences comprised residues 82–87, as under-lined in the alignment of c- and b-type crystallins (Table 1.) However, when the latter experiment was carried out [47]

by replacing the linker of the b-type protein with a longer c-type peptide sequence that included two extra residues at the N-terminus (Pro80 and Ile81 in the alignment above), the engineered b-type protein did become monomeric.Thus, if the linker peptide connecting motifs M2 and M3 of the protein is defined as the sequence comprising residues 80–87 [20], the linker sequence does appear to have a role as a determinant of the dimeric structure.It must be noted that the Pro residue at position 80 is strictly conserved in b-type crystallins, whereas in c-type proteins a Leu is found at that position (with the single exception of a Ser in cA-crystallin) This suggests that the presence of a Pro at position 80 can force the linker into an extended conformation, that typical

of b-type crystallins, which does not allow for a sufficiently high local concentration of interdomain interacting residues [23].In the absence of Pro80, these residues can interact and the two domains associate into a c-type monomer.It is tempting to propose that a key amino-acid substitution (a Ôprimary mutationÕ) in the evolution of c-type and b-type crystallins from their common ancestor was the insertion of

a Pro residues at that position in the b-type sequences, and

of a hydrophobic residue in c-type crystallins

Contrasting results were obtained in another laboratory, showing that a recombinant b-crystallin variant is isolated

as a dimer also when its linker is replaced with a c-type linker [57].Although the b-crystallin used in the latter experiment was rat b-B3, instead of bovine b-B2, and the replaced fragment was two residues longer, the replacing linker was from the same c-B crystallin as in the experiment cited above [46].The insertion of a C-terminal Tyr residue in the substituting fragment, and the presence of a Ser instead

of a Thr, may explain the contrasting results.If these were both confirmed, we may only surmise that in these types of engineering experiments only limited areas of the protein structure under test are narrowly illuminated, while other effects of the engineering on other areas of the protein structure remain in the dark, and may affect the interpret-ation

However, the overall conclusion that the linker peptides did have a role in the evolution of monomeric vs.oligomeric crystallins is convincing.In this respect, it would be interesting to determine the structure of the crystallin-type protein from the sponge gene [36], in which a short (only three residues) interdomain linker peptide has been identi-fied, i.e with a length typical of c-type crystallins linkers

As for the terminal extensions, they are mostly flexible and mobile [58] and do not seem to play any roles in folding and domain association [44,59,60].The proximal stretch of

Table 1 Alignment of c- and b-type crystallins The exchanged sequences comprise residues 82–87 (underlined).

bB2 crystallin linker P I K V D S Q E

cB crystallin linker L I P Q H T G T

the C-terminal extension in the b-type structure instead is

not flexible, and has been suggested to mimic a noncovalent

interdomain linker because it introduces its Trp175 residue

in a hydrophobic pocket on the surface of the N-domain

from the partner subunit [20].When the whole C-terminal

extension, including Trp175, is removed, b-type crystallin

can still associate into dimers and tetramers [47]

But the terminal extensions, although apparently not a

determinant in the structural chemistry of present-day

crystallins, may have instead had key roles in the

evolu-tionary modular assembly of these proteins.It has been

found that although isolated, recombinant c-type

C-domains cannot associate into noncovalent structures to

mimic a c-type crystallin [7], yet they will associate after the

removal of the terminal Tyr residue from their C-terminal

extensions.In the 3D structure of this des-Tyr-c-C-domain,

the C-terminal extension hinders the association of the two

domains by interacting with the hydrophobic interdomain

interface.This destabilizing effect would not be exerted

when the covalent interdomain linker is in position and

displaces the peptide extension out into the solvent.These

results suggest that the extended form of the linker peptide,

characteristic of b-crystallins, could have evolved directly

from the C-terminal extension of a two-domain ancestor [7]

An independent experimental approach has led to similar

conclusions.The C-terminus of the C-domain extension of

rat bB2 crystallin has been fused by protein engineering with

the N-terminus of the N-domain from the partner subunit

[50].Because the engineering also discontinued the

interdo-main linkers in both subunits, a circularly permuted

structure was obtained.In this structure, the C-terminal

extension was turned into an interdomain linker.The

resulting expressed protein was still a dimer, but differed

from the wild-type bB2 dimer, in that its domain pairing

was that typical of c-crystallin

These experiments support the proposal that the

exten-sions may have been the evolutionary precursors of

interdomain linkers, but also confirm the crucial role played

in evolution by the linkers themselves.They hint at the

possibility that circular permutation may have been one of

the mechanisms employed in the evolution of new crystallin

structures [15,49].In modular constructions, structural

variation depends on the different ways modules are

assembled, i.e on the different types of structural elements

connecting and pairing the modules.Domain extensions

could well have been exploited by evolution to generate a

variety of linkers in order to get the creative advantages

inherent in modular assemblies

It has been proposed that another experimental approach

to obtain insight to the evolutionary history of an

oligomeric protein is to investigate its unfolding/refolding

[3,8].This is based on the idea that the folding pathway of

an oligomer might reiterate its evolutionary pathway.Thus,

it may be of interest to analyze the results of unfolding/

refolding experiments carried out on crystallin-type

pro-teins

Spherulin 3a [61], the single-domain crystallin-type

pro-tein, unfolds in a highly cooperative fashion with a two-state

transition [2,62,63].Two-domain proteins, such as protein S

[64] and a c-type crystallin [45], unfold instead with

three-state transitions, just as a b-type crystallin does [51].It

should be added that the isolated N- or C-domains,

prepared by recombinant technology unfold cooperatively with two-state transitions [54]

The intermediates in the unfolding pathway of both protein S and c-type crystallin have been described as presenting a still folded N-domain and a fully unfolded C-domain.In contrast, in the unfolding pathway of b-type crystallin the N-domain unfolds first while the C-domain remains folded.Interestingly, the isolated b-type C-domains are monomeric, whereas isolated N-domains associate Based on these results, and on the findings described above, we can envisage that single-domain crystallin-type proteins natural as Spherulin 3a, or artificially produced as the isolated domains from c- and b-type crystallins resemble the evolutionary ancestors of two-domain crystallin.Hence,

we may regard these one-domain proteins as stable mono-mers.Once rendered unstable through mutations in their encoding genes, they could find a new stable conformation only upon gene fusion leading to domain association.This evidently happened along distinct, parallel evolutionary paths, for c-type and protein-S crystallin-type proteins, and

b)type crystallins, respectively.Thus, the results of the unfolding/refolding experiments and their interpretation are

in support of the evolutionary pathway illustrated in Fig.3

C O N C L U S I O N S

It appears that the findings described above, based on structural and protein engineering studies or on molecular genetics analyses, lead to the same conclusions.Both sets of data indicate that a series of gene alterations and fusions led from crystallin ancestors coding for proteins made up of a single Greek-key motif to two-motif/one-domain proteins,

to two-domain c-type crystallin monomers, or two-domain/ two-monomer b-type dimers.A key role in the evolutionary cascade was apparently played by the gene sequences encoding the C-terminal extensions downstream to the motif encoding exons in one-motif and one-domain ances-tors.These are the sequences involved in the gene fusion molecular events and especially marked by high substitution rates.In the present day, postfusion two-domain crystallin genes, homologous sequences encode the interdomain linker peptides.These DNA sequences were the hot spots in the ancestral crystallin genes, where evolution intensely experi-mented to generate protein sequences that independently evolved into two distinct paths, leading to different linker conformations for c- and b-crystallins, hence to monomers and dimers, respectively

A C K N O W L E D G E M E N T S

I am grateful for comments and criticism on the manuscript to J.F Riordan (Harvard Medical School), G.Wistow (NIH), M.Riley (MBL, Woods Hole), and my colleagues in Naples: M.V.Cubellis, A.Di Maro, T.Giancola, R.Piccoli, and A.Russo.The rendering of molecular graphics for Fig.1 was provided by M.V Cubellis Figures 2 and 3 were drawn by A.Di Maro; I am very grateful to both.

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