REVIEW ARTICLE
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 oligomeric 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,

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

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.
STRUCTURAL FEATURES OF
MONOMERIC AND DIMERIC
CRYSTALLINS
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-andb-crystallins,
andsimplyreferredtoasc-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

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.
MOLECULAR GENETICS STUDIES:
FACTS AND HYPOTHESES
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

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-andc-type nota-
tions indicate motif arrangements in: spherulin 3a, Streptomyces
protease inhibitor, S-protein-, G. Cydonium protein, b-andc-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 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.
3124 G. D’Alessio (Eur. J. Biochem. 269) Ó FEBS 2002
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.

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

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
Ó FEBS 2002 On the evolution of crystallins (Eur. J. Biochem. 269) 3125

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
domainswap[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.
STRUCTURAL STUDIES: FACTS
AND HYPOTHESES
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

(thick bars) introns; the lack of separation
lines between motifs or domains indicate that
inthosecasesthepresenceorabsenceof
introns has not been determined.
3126 G. D’Alessio (Eur. J. Biochem. 269) Ó FEBS 2002
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

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

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).
80 87
bB2 crystallin linker PIKVDSQE
cB crystallin linker LIPQHTGT
Ó FEBS 2002 On the evolution of crystallins (Eur. J. Biochem. 269) 3127
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

[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-andb-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

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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