Abstract
The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW–PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.
Highlights
Modular multidomain proteins are common cell-cycle regulators in eukaryotes [1, 2]
Our paramagnetic relaxation enhancement (PRE) experiments show that the transient interdomain contacts in apo-Pin1 exceed the range previously suggested
PREs show reduced interdomain contact upon binding of pCdc25C to the WW domain, which involves increased domain separation concomitant with intra-WW domain conformational shifts consistent with those induced by binding of pS/ T-P substrates [13, 29, 30]
Summary
Modular multidomain proteins are common cell-cycle regulators in eukaryotes [1, 2] Their mechanisms often depend on transient interactions between domains serving complementary functions. Pin consists of an N-terminal WW domain (residues 1–39) that is linked by a flexible tether to a larger C-terminal PPIase domain (residues 53–163) (Fig. 1) Both domains have sites for specific pS/T-P recognition. Previous studies of Pin have documented changes in PPIase activity caused by remote perturbations in the WW domain that include substitution mutations [10,11,12,13] and post-translational modifications [10, 14] These long-range effects indicate the presence of a mechanism for interdomain cross-talk that remains the subject of active investigation. The transience is a consequence of the extensive relative motion between the two domains afforded by the flexible intervening linker (residues 40–52) [16,17,18]
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