Biology Reference
In-Depth Information
contribute to factor X binding. By contrast, mutations at factor IX residues which
are sequence conserved in mammals and one or two other serine proteases were
found to cluster in the serine protease domain and also at domain boundaries
within the protein structure. Presumably, docking of the constituent domains of
factor IX and the other homologous serine proteases during protein folding
requires amino acid conservation at these boundaries. Finally, factor IX residues
which are sequence conserved between mammals but not between other serine
proteases may be exclusively important for the structure and function of factor
IX. When the 3D structure of the factor IX protein was considered, spatially clus-
tered groups of mutations at such residues became apparent on the surface of the
EGF domains, in regions implicated in the binding of factors Va and VIIIa, and
of the serine protease domain.
Regions which exhibit similar functions in different homologous proteins may
not only be sequence conserved but may also exhibit structural conservation
(Greer, 1990). Although
sequence conserved regions
(SeqCRs) of mammalian serine
proteases are invariably structurally conserved,
structurally conserved regions
(SCRs) may differ markedly with respect to their amino acid sequences. SCRs
were defined by Greer (1990) as those portions of known protein structures that
overlap very well when superimposed. In serine proteases, SCRs usually comprise
secondary structure elements, the active site and other essential structural frame-
work residues of the molecule.
The locations of structurally conserved regions in human factor IXa were
determined by Wacey
et al
. (1994) employing their homology model of the factor
IX protein. No clear relationship was noted between the severity of the hemo-
philia B phenotype and the level of structural conservation of a mutated factor IX
amino acid residue, although substitutions at structurally conserved residues
were estimated to have an approximately two-fold higher likelihood of resulting
in a disease state than mutations at nonconserved residues. Interestingly, mutated
sites which were not sequence-conserved were nearly all structurally conserved.
The only exception involved two missense mutations at Gly59. However, Gly59
lies immediately adjacent to a type
hairpin SCR and would be predicted to be
critical in defining this structural element (Swindells and Thornton, 1991). Some
SCRs, although not sequence-conserved, may thus serve as structural supports
through their backbone interactions and should therefore be regarded as 'scaf-
folding' residues rather than 'spacers' (Bottema
et al.
, 1991).
The topological properties of a mutated factor IX amino acid residue are also
important for determining clinical severity. Mutations at residues with their side
chains pointing away from the solvent ('buried residues') were found to cause
severe hemophilia 1.5 times more often than mutations at residues with solvent-
accessible side chains (Wacey
et al.
, 1994). Finally, the likelihood of a mutation
resulting in a severe disease phenotype was higher for substitutions in hydropho-
bic as opposed to polar regions, probably because of the critical importance of
these residues for correct protein folding (Kragelund
et al
., 1999). This is consis-
tent with the conclusions of other workers that amino acid substitutions occur-
ring in the protein core give rise to a 'continuum of increasingly non-native
properties' affecting the stability and/or the folding dynamics of the protein
(Alber, 1989; Lim
et al
., 1992; Pakula and Sauer, 1989).
