Discussion
In summary, we have collected evidence that support the view that the
residues required to sustain a promiscuous reaction in serum albumin
could have evolved to hold a biological function. We have found that
those residues involved in the reaction (Lys 199 and Arg 222 in HSA) are
located in a large and hydrophobic cavity and are conserved during
evolution (Figure 2) Interestingly, the pka shifts in these amino acids
are also conserved to produce polarized “acid” and “basic” residues
independently of being Lys or Arg. As we previously showed, the swapping
in the Lys/Arg (HSA) and Arg/Lys (BSA) key residues (Table 1) in the
natural sequences analyzed is statistically different from the
occurrence by chance of the simple replacements of Lys and Arg.
Evolutionary models as those used in this work (see Methods) consider
residue replacements during evolution in a site-independent fashion.
However, conservation of pka shifts in species also indicates
physicochemical environmental constraints indicating a putative
functional adaptation. Another piece of evidence is the finding of a
common tunnel in the three structures analyzed. Tunnels allow the
transit of ligands between cavities and protein surfaces. As shown in
Figure 4, three important residues lie at the bottom of the tunnel (Lys
199, Arg 218, and Arg 222 in HSA numbering). Among these, Lys 199 and
Arg 222 were previously suggested to be involved in promiscuous
reactions41,48,49.
Also, Arg 218 is 100% conserved in serum albumins and its replacement
has been related to the occurrence of human diseases. Replacement of Arg
218 to His or Pro produces abnormal albumin with increased affinity for
serum thyroxine found in an autosomal dominant condition called Familial
dysalbuminemic hyperthyroxinemia50,51.
This condition is caused by an abnormal albumin molecule with an
increased affinity for the hormone thyroxine. Supporting the putative
role of this tunnel is the fact that two flanking residues (Arg 222 and
His 440 in HSA see Figure 4) are evolving under positive selection.
Our results offer a biological interpretation of the observed
promiscuous catalytic activity in serum albumins. In addition, a new
perspective is offered in the classification of albumins as possible
enzymes, in light of the observation of the selective pressure in the
key positions for catalysis, typically observed during functional
divergence52.
From this point of view, the catalytic properties of the serum albumins
documented in the present work could be involved in an unknown
biological process rather than a promiscuous behavior. These results not
only allow us to better characterize the family but also open up
interesting questions about the origin of promiscuous behavior and the
evolution of protein function.