Description

High-affinity electrostatic binding of the noble gas krypton to FGF: implications & possibilities

Ogan Gurel
30 November 1993

Experimental
Krypton was introduced at approximately 5 atm into a quartz capillary containing a single triclinic bFGF crystal. Data was collected to 2.0A resolution with a cumulative Rmerge of 3.3% and an overall completeness of 80%. Native data was also collected (78% complete, 8% Rmerge to 2.0A resolution) under identical crystallization, data collection and data reduction procedures.
Difference fouriers were calculated using a refined model (without waters) of hi-MW bFGF for phases. A high-resolution (2A) difference fourier map showed a single strong peak at a height of 356 located near the surface of the protein in the vicinity of residues 77 and 78. Closely flanking this peak are two smaller, distinct side peaks at 106 disposed such that all three peaks lie along a line perpendicular to the surface of the protein between the side chains of glutamate 78 and lysine 77.
Structure of the binding site
The krypton atom is closely packed between the positively charged NZ of lysine 77 and the negatively charged 0E2 of glutamate 78 at a distance of 4.5 A between atomic centers. The fact that this peak lies between a positive and a negative charge is strong evidence against a "conventional" anion or cation in the site. Additional coordination appears to be provided by the positively charged NH2 of arginine 81 and the negatively charged 0E1 of glutamate 91 but these are at longer distances of 5.8 and 4.9 A respectively.
There is no observable water at the krypton site both in native Fo-Fc maps as well as in other published FGF studies. There is, however, a high B-value water located somewhat below the site close to glutamates 77 and 91. The Fo-Fo map shows a distinct negative peak overlying the water site indicating that it might have been pushed out during the binding of krypton although this appears not to be obligatory in steric terms.
We had originally anticipated that the binding site would be hydrophobic in nature. The result we have here of an almost purely electrostatic interaction is both unexpected and unprecedented.

Noble gas chemistry
Very few noble gas compounds or complexes are known and those that are have been isolated using powerful reagents and under extreme conditions of temperature and pressure. The krypton site here is completely stable at room temperature without the benefit of any extraordinary pressures. The existence of such a complex speaks strongly about the almost incredible specificity and affinity of binding between proteins and their ligands. It is all the more amazing when one considers that krypton is spherically symmetric and uncharged -- characteristics that don't immediately lend themselves to any special specificity.
Most noble gas chemistry involves the oxidation of xenon (or more rarely, krypton) by powerful oxidizing agents such as PtF6. In addition, the noble gases with their electron rich valence shells have been classified as extremely weak Lewis bases. In chemical terms, the krypton-FGF coordination follows the latter pattern with electrons being "donated" to the electrophilic proton on NZ of lysine 77 which is facilitated by the polarizing effect of 0E2 on glutamate 78.
Thermodynamics of binding
A very rough calculation of the enthalpy of condensation and the entropy of a monatomic gas indicate that the minimum free energy of binding must be -11 kcal/mol. Additional binding energy must also be devoted to polarizing the krypton and to maintaining the site at nearly 100% occupancy This leads to free energies of binding that are on the order of -15 to -20 kcal/mol. All of these are very gross numbers and need to be refined with a better understanding of the structure of the site. Nevertheless, it is clear that the binding of the protein to this symmetric, uncharged atom is both strong and specific. The krypton site represents a unique opportunity