Relative stability of the open and closed conformations of the active site loop of streptavidin

Abstract

The eight-residue surface loop, 45-52 (Ser, Ala, Val, Gly, Asn, Ala, Glu, Ser), of the homotetrameric protein streptavidin has a "closed" conformation in the streptavidin-biotin complex, where the corresponding binding affinity is one of the strongest found in nature ( G ∼ -18 kcal/mol). However, in most of the crystal structures of apo (unbound) streptavidin, the loop conformation is "open" and typically exhibits partial disorder and high B-factors. Thus, it is plausible to assume that the loop structure is changed from open to closed upon binding of biotin, and the corresponding difference in free energy, F = F open -F closed in the unbound protein, should therefore be considered in the total absolute free energy of binding. F (which has generally been neglected) is calculated here using our "hypothetical scanning molecular-dynamics" (HSMD) method. We use a protein model in which only the atoms closest to the loop are considered (the "template") and they are fixed in the x-ray coordinates of the free protein; the x-ray conformation of the closed loop is attached to the same (unbound) template and both systems are capped with the same sphere of TIP3P water. Using the force field of the assisted model building with energy refinement (AMBER), we carry out two separate MD simulations (at temperature T = 300 K), starting from the open and closed conformations, where only the atoms of the loop and water are allowed to move (the template-water and template-loop interactions are considered). The absolute F open and F closed (of loop + water) are calculated from these trajectories, where the loop and water contributions are obtained by HSMD and a thermodynamic integration (TI) process, respectively. The combined HSMD-TI procedure leads to total (loop + water) F = −27.1 ± 2.0 kcal/mol, where the entropy T S constitutes 34% of F, meaning that the effect of S is significant and should not be ignored. Also, S is positive, in accord with the high flexibility of the open loop observed in crystal structures, while the energy E is unexpectedly negative, thus also adding to the stability of the open loop. The loop and the 250 capped water molecules are the largest system studied thus far, which constitutes a test for the efficiency of HSMD-TI; this efficiency and technical issues related to the implementation of the method are also discussed. Finally, the result for F is a prediction that will be considered in the calculation of the absolute free energy of binding of biotin to streptavidin, which constitutes our next project.

Calculation of the Absolute Free Energy of Binding and Related Entropies with the HSMD-TI Method: The FKBP12-L8 Complex

“…It should be noted that the angles (α k ) are calculated in radians, which can lead to negative entropies (in contrast to our previous studies where using degrees led to positive entropies 22,25-29 ); this is not unexpected as $S_{\mathrm{ligand}}^{\mathrm{normalA}}$ is defined up to an additive constant and we are interested only in entropy differences Δ S ligand (eq 8) where this constant cancels out. As expected by theory, the results for $S_{\mathrm{ligand}}^{\mathrm{normalA}}$ decrease systematically as the approximation improves, i.e., with increasing n f , but they remain unchanged as a function of δ; however, these results have not converged even for n f =6000.…”

The F130L mutation in streptavidin reduces its binding affinity to biotin through electronic polarization effect

“…BTN binding is accompanied by the stabilization of a flexible loop (Ser45 to Ser52) connecting β strands 3 and 4 (L3/4), which closes after the biotin binds, acting like a “lid” over the binding pocket (see Fig. ) and contributing to the extremely slow biotin dissociation rate . A wealth of theoretical and computational investigations has been devoted to this system to unveil the binding mechanism.…”

Entropy and Free Energy of a Mobile Loop Based on the Crystal Structures of the Free and Bound Proteins

“…However, this is not a failure of HSMD-TI per se but reflects the uncertainty in the crystal structure of the bound protein. One should also bear in mind that no other studies of a loop attached to two templates have been carried out thus far and besides our HSMD-TI papers [8–10,62] only few calculations of Δ F loop (based on a single template) are available [48,49]. Thus, the present work should be considered as the first step and a basis for future more extensive studies, which can be pursued in several avenues: for example, HSMD-TI can be applied to a mobile loop with known Δ F loop where the quality of the crystal structures of both, the bound and the free protein is high.…”

“…The conclusion is that to recover the experimental free energy difference the water density should be close to that of bulk water, and the minimum template size is of 1,000 atoms for this loop. In all these studies and in a recent study of a mobile loop of the protein streptavidin [62] the free and bound loop structures were attached to a common template that was “cut” from the crystal structure of the free loop. Obviously, one would seek to treat each loop attached to its own template.…”