Molecular Mechanisms of Tight Binding through Fuzzy Interactions

Abstract

Many intrinsically disordered proteins (IDPs) form fuzzy complexes upon binding to their targets. Although many IDPs are weakly bound in fuzzy complexes, some IDPs form high-affinity complexes. One example is the nonstructural protein 1 (NS1) of the 1918 Spanish influenza A virus, which hijacks cellular CRKII through the strong binding affinity (K ∼10 nM) of its proline-rich motif (PRM) to the N-terminal Src-homology 3 domain of CRKII. However, its molecular mechanism remains elusive. Here, we examine the interplay between structural disorder of a bound PRM and its long-range electrostatic interactions. Using x-ray crystallography and NMR spectroscopy, we found that PRM retains substantial conformational flexibility in the bound state. Moreover, molecular dynamics simulations showed that structural disorder of the bound PRM increases the number of electrostatic interactions and decreases the mean distances between the positively charged residues in PRM and the acidic residues in the N-terminal Src-homology 3 domain. These results are analyzed using a polyelectrostatic model. Our results provide an insight into the molecular recognition mechanism for a high-affinity fuzzy complex.

Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

“…Moreover, Wu et al identified that the electron density of the C-terminal region of a PRM peptide is missing in the crystal structure of its complex with nSH3 CRK [48], indicating high conformational flexibility of the region. In line with these findings, our previous studies indicated that a fuzzy electrostatic interaction between the structurally dynamic PRM 1918 peptide and the isolated nSH3 CRK domain drives high affinity binding [31,39]. The fuzzy electrostatic interaction Figure 5.…”

“…To test the hypothesis, we measured the KD values between CRK-II and the 1918 NS1 ED-CTT :p85 iSH2 complex in the presence of 1M NaCl. The fuzzy electrostatic interaction, in essence, is a long-range electrostatic interaction mediated by a conformationally flexible ligand [49]; thus, the interaction is screened in a high ionic strength solution [39]. Indeed, we observed that the KD value increased by more than 24-fold in the presence of 1M NaCl (Figure 6B), compared to that in 100 mM NaCl ( Figure 4B).…”

“…In contrast, truncating the CTT (1918 NS1 ED-∆CTT ) abolished binding to CRK ( Figure 2D), indicating that the 1918 NS1:CRK interaction is mainly mediated by the PRM in the CTT. The isolated PRM peptide is considered to be structurally disordered and lacks higher order structures [31,39]. However, the conformation of the CTT in 1918 NS1 was not directly characterized; it thus remains uncertain whether the PRM region is indeed structurally disordered.…”

“…Although our binding data suggested that the PRM might be exposed to solvent in the context of 1918 NS1 ED-CTT , it is not direct evidence of high conformational flexibility of the PRM and overall CTT in the protein. We showed previously that the conformational flexibility of the PRM 1918NS1 peptide plays a critical role in increasing its affinity to the nSH3 domain of CRK [31,39]. Thus, to understand the binding mechanism between 1918 NS1 and CRK, the conformational flexibility of the CTT within 1918 NS1 ED-CTT should be examined.…”

What Are We Missing by Not Measuring the Net Charge of Proteins?

“…[1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering. [51][52][53][54][55][56] It is tempting to assume that the formal net chargeo faprotein predicted from generalized residue pK a values (Z seq )issosimilar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about ap rotein's net charge.…”