Direct Observation of the Dynamic Process Underlying Allosteric Signal Transmission

Abstract

Allosteric regulation is an effective mechanism of control in biological processes. In allosteric proteins a signal originating at one site in the molecule is communicated through the protein structure to trigger a specific response at a remote site. Using NMR relaxation dispersion techniques we directly observe the dynamic process through which the KIX domain of CREB binding protein communicates allosteric information between binding sites. KIX mediates cooperativity between pairs of transcription factors through binding to two distinct interaction surfaces in an allosteric manner. We show that binding the activation domain of the mixed lineage leukemia (MLL) transcription factor to KIX induces a redistribution of the relative populations of KIX conformations toward a high-energy state in which the allosterically activated second binding site is already preformed, consistent with the Monod-Wyman-Changeux (WMC) model of allostery. The structural rearrangement process that links the two conformers and by which allosteric information is communicated occurs with a time constant of 3 ms at 27 degrees C. Our dynamic NMR data reveal that an evolutionarily conserved network of hydrophobic amino acids constitutes the pathway through which information is transmitted.

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…4). A similar cooperative mechanism is found in the KIX-MLL-cMyb heterotrimer 45 , in which a two-fold enhancement was linked to an MLL-induced enrichment of the population of the KIX conformer, which has high affinity for cMyb 46 .…”

Prepaying the entropic cost for allosteric regulation in KIX

“…Previous studies have observed changes in the KIX structure resulting from transcription factor binding, and both MLL and c-Myb have been shown to allosterically affect the affinity of KIX for the complementary peptide (4,20,(28)(29)(30)(31)(32). However, a connection between the changes in structural dynamics in KIX and how this directly leads to shifts in transcription factor binding affinities remain tenuous.…”

“…The binding of either MLL or c-Myb to one of these promiscuous sites can stabilize the C terminus of the α 3 helix of KIX and/or decrease the mobility of the L 12 -G 2 loop (4,20,(28)(29)(30)(31)(32), but the functional role of these changes remains to be delineated. Furthermore, in vitro, KIX alone demonstrates a ∼2-fold lower affinity for c-Myb and ∼1.6-fold lower affinity for MLL compared with KIX bound to MLL or c-Myb, respectively (13).…”

NMR reveals novel mechanisms of protein activity regulation

“…[33][34][35] Numerous examples are currently available highlighting the inextricable link between protein dynamics and allostery 8,36 . Long-range allosteric coupling between sites through changes in internal dynamics were seen between the nucleotide-binding cleft and the preprotein-binding site in SecA ATPase, 37 between the coordination Na þ site and exosite I in thrombin that regulates enzyme's specificity, 38 between the substrate-binding site and distal loop in dihydrofolate reductase, 39 between the mixed lineage leukemia)-and c-Myb binding sites of the KIX domain of CREB-binding protein, 40 in PDZ signaling domains, 41 the serine protease inhibitor eglin c, 42 upon binding of barstar to the RNase barnase, 43 in the interaction between the Rho GTPasebinding domain and Rac1, 44 upon cyclic nucleotide binding to the exchange protein activated by cAMP, 45,46 a in V-type allosteric enzyme, 47 and in protein kinase A, 48,49 only to mention few recent examples from a long list of systems characterized over the years. Dynamic changes in these systems were accompanied by varying extent of structural changes, ranging from minimal to substantial.…”

“…55 Determining the chemical shifts of the excited state using relaxation dispersion have in several cases provided information that those states structurally resemble related functional states. 40,53,82,83,87 Incorporation of chemical shifts in NMR structure refinement and the development of new NMR experiments that can report on internuclear vector orientations in excited conformational states provide valuable structural constraints that may enable direct structural characterization of higher energy protein conformations. [88][89][90] Kay and coworkers recently reported the structure of an ''invisible'' protein folding intermediate, a remarkable achievement convincingly showing that structure determination of weakly populated conformational states are within reach.…”