High-resolution x-ray crystallographic

structural analysis of hi-MW bFGF:

the role of dynamic flexibility in protein function
Ogan Gurel
9 March 1994

Growth   factors   are   important   in   both   normal   physiology   and   disease.     The   especially 
ubiquitous   fibroblast   growth   factors   participate   in   tissue   repair   and   angiogenesis   by 
stimulating the proliferation of a wide spectrum of mesodermally and neuroectodermally­
derived   cells.     Understanding   the   structural   biology   of   these   growth   factors   is   crucial   to 
understanding   their   function   and 
dysfunction.  Here, we report the three­
dimensional,   high­resolution   1.7Å 
structure  of   an   N­terminally   extended, 
hi­MW   species   of   basic   fibroblast 
growth   factor   (bFGF).     The   overall 
structure is that of a β­trefoil (see figure) 
— a fold which is shared by other FGFs 
as   well   as   by   interleukin–1.       Features 
specific   to   this   new   structure 
demonstrate   that   the   N­terminal 
extension — which has been implicated 
in   the   differential   subcellular 
localization   of   bFGF   —   is   structurally 
disordered.  These results imply that the 
biophysical basis of this protein sorting 
process   must   be   more   subtle   than   the 
simple   recognition   of   a   static 
conformation.     Dynamic   flexibility   clearly   plays   a   role.     Our   FGF   has   also   been   post­
translationally modified by disulfide linkage to two exogenously added cysteine molecules 
which function to protect bFGF against oxidative damage  in vivo.   The structure of one of 
these cysteines is clearly defined as part of a lattice contact while the other is structurally 
disordered.   From this we conclude that there is no intrinsic structure to this novel post­
translational modification and that dynamic flexibility may also modulate the structural basis 
of oxidative protection.  In addition to the novel structural findings, this work also reports on 
important x­ray crystallographic methodological advances.   In particular, we discuss a  new  
cell reduction scheme for triclinic space groups, the use of anisotropic temperature factors during 
structure refinement to improve the accuracy of the model and more precisely describe the 
dynamic motions of the protein and finally the application of a bulk solvent mask that further 
improves the refined model particularly at surface residues that play a crucial role in receptor 
binding and biological function.  These studies allow us to dissect not only the static but also 
the  dynamic  structure   of   FGF   to   unprecedented   detail.     In   this   context   we   discuss   the 
implications  of   the   structure   for   receptor­binding   and   rational   structure­based   drug 
design.