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provide further evidence of what must have been a very active phase in the evolu-
tion of the modern vertebrate genome.
10.2.4 Reconstruction of a common ancestor of the vitamin K-dependent
coagulation factors
The evolutionary scenario depicted in
Figure 10.1
was based solely upon the
highly conserved regions of the reconstructed ancestral mammalian cDNA
sequences. However, this phylogeny was used by Krawczak
et al
. (1996) in an
attempt to reconstruct the common ancestor of all five vitamin K-dependent fac-
tors by means of molecular modelling. To this end, the less well conserved regions
of the mammalian ancestral sequences were first aligned in the order stipulated by
the phylogeny. The alignment was then used to determine the nucleotides present
at each node of the phylogeny, moving upwards through the tree to its root. The
resulting cDNA sequence, representing the putative common ancestor of all five
vitamin K-dependent factors, contained several gaps which yielded ambiguities
in the sequence of the ancestral protein. However, since the protein core had to
contain a full complement of residues for meaningful molecular modelling to be
possible, all such amino acids were replaced with the analogous residue found in
extant human thrombin as a 'best guess'.
One way to examine the plausibility of the deduced amino acid sequence of the
vitamin K-dependent factor ancestral protein would have been to express it
in
vitro
and to characterize it biochemically (Malcolm
et al
., 1990; Shih
et al
., 1993).
An alternative strategy was to construct a model of the tertiary structure of the
protein by comparative methods and examine its topology and biophysical prop-
erties. Such a model was created by Krawczak
et al
. (1996) using the X-ray crys-
tallographic coordinates of the heavy chain of
-thrombin as a template. This
template was aligned against the high resolution structure of seven other serine
proteases and the amino acid sequence of the reconstructed ancestral protein
(Greer, 1990). Sequence modifications became necessary in this process at two
variable regions, which were longer than the analogous loops present in extant
serine proteases, and a single unpaired Cys residue (Cys22), which was replaced
by Ala as in extant human thrombin. Extant human thrombin was chosen for
comparison in this and other instances since, of all of the modern hemostatic ser-
ine proteases, it bore the strongest sequence homology to the ancestral protein.
Moreover, it was assumed (Doolittle, 1993) that the vitamin K-dependent factor
ancestral protein would have been capable of performing the end effector function
of thrombin in the hemostatic cascade, that is the cleavage of soluble fibrinogen
to generate insoluble fibrin clot.
The putative ancestral protein was found to contain 86 charged groups at pH
7.0, very similar to the number (89) found in extant human thrombin. In both
cases, the great majority of these charges were noted to be accessible to water. The
global electrostatic distribution across the ancestral protein's surface was rela-
tively uniform by comparison with the dipolar distribution evident in extant
human thrombin. Both structures still possessed an unbroken equatorial belt of
negative charge but the extent of the electrostatic field strength in the ancient
protein was not as strong as that of extant thrombin. It was speculated that the
