Domain cooperativity in multidomain proteins: what can we learn from molecular alignment in anisotropic media?

Abstract

Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, tandem SH3 efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. To test the accuracy of theoretical predictions, we prepared a number of tandem-SH3 samples in PEG/hexanol alignment media. These samples were used to measure backbone 1HN-15N residual dipolar couplings associated with the two globular domains within the tandem. The experimental alignment tensors determined in this fashion were compared with the results of the PALES-based simulations, broadly confirming the anticipated trends. The results of the comparison, however, fall short of quantitative agreement. In particular, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a measure of domain-domain cooperativity, thus identifying the situations where two domains transiently interact with each other or become coupled through a partially structured linker.

Insights into the allosteric regulation of Syk association with receptor ITAM, a multi-state equilibrium

“…The (GGS) 3 GS(GGS) 3 linker of tSH2 FX was shown to be flexible and enables independent rotation of the connected globular domains. 28 …”

Examination of Matrix Metalloproteinase-1 in Solution

“…Although detailed structural characterization of the bound-state conformation is often possible, much more difficult is the analysis of the conformations sampled by multidomain proteins before the interaction. However, analysis of the conformational space experienced by the free protein is useful not only to investigate the mechanism of binding, but also to determine the role of the different domains in the identification of substrates or partners, to predict new possible substrates or partners, and to investigate natural and new mechanisms of inhibition (3,22,24,29,35,(57)(58)(59)(60).…”

Interdomain Dynamics Explored by Paramagnetic NMR

“…This requires accurate modeling as has been recently pointed out again. 25 Using residual dipolar couplings from alignment media has recently been shown to deliver biased results on the example of the small biomolecules lactose and chitobiose. In these examples, RDCs derived from measurements in alignment media 26 did not reproduce the ensembles derived from paramagnetic alignment 27,28 which most reliably detects domain motion as described below.…”