Allostery is a fundamental mechanism of regulation in biology. The residues at the end points of long-range allosteric perturbations are commonly identified by the comparative analyses of structures and dynamics in apo and effector-bound states. However, the networks of interactions mediating the propagation of allosteric signals between the end points often remain elusive. Here we show that the covariance analysis of NMR chemical shift changes caused by a set of covalently modified analogs of the allosteric effector (i.e., agonists and antagonists) reveals extended networks of coupled residues. Unexpectedly, such networks reach not only sites subject to effector-dependent structural variations, but also regions that are controlled by dynamically driven allostery. In these regions the allosteric signal is propagated mainly by dynamic rather than structural modulations, which result in subtle but highly correlated chemical shift variations. The proposed chemical shift covariance analysis (CHESCA) identifies interresidue correlations based on the combination of agglomerative clustering (AC) and singular value decomposition (SVD). AC results in dendrograms that define functional clusters of coupled residues, while SVD generates score plots that provide a residue-specific dissection of the contributions to binding and allostery. The CHESCA approach was validated by applying it to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC) and the CHESCA results are in full agreement with independent mutational data on EPAC activation. Overall, CHESCA is a generally applicable method that utilizes a selected chemical library of effector analogs to quantitatively decode the binding and allosteric information content embedded in chemical shift changes. L ong-range allosteric perturbations are propagated not only by structural changes but also by effector-dependent modulations in dynamics (1-23). The end points of these long-range allosteric signal propagations are effectively characterized by the comparative analysis of the structural and dynamic profiles of apo and effector-bound states (2, 7). However, what remains experimentally challenging is often defining the networks of residues that mediate the cross-talk between distal sites. Such clusters of coupled residues are particularly elusive in allosteric processes with a significant dynamically driven component (11-17), as in this case the allosteric signal propagation relies on subtle, but critical, conformational and side-chain packing rearrangements that often fall below the resolution of common X-ray or NMR structure determination methods (2, 7, 24).Here we introduce a general experimental method to map allosteric networks based on the covariance analysis of NMR chemical shifts. The chemical shift covariance analysis (CHESCA) is based on two simple but general notions. The first assumption is that the subtle but functionally relevant structural changes that underlie the allosteric modulations of dynamics are effectively probed by accurately ...

Protein kinase A (PKA) is a prototype of multidomain signaling proteins functioning as allosteric conformational switches. Allosteric transitions have been the subject of extensive structural and dynamic investigations focusing mainly on folded domains. However, the current understanding of the allosteric role of partially unstructured linkers flanking globular domains is limited. Here, we show that a dynamic linker in the regulatory subunit (R) of PKA serves not only as a passive covalent thread, but also as an active allosteric element that controls activation of the kinase subunit (C) by tuning the inhibitory preequilibrium of a minimally populated intermediate (apo R). Apo R samples both C-binding competent (inactive) and incompetent (active) conformations within a nearly degenerate freeenergy landscape and such degeneracy maximally amplifies the response to weak (∼2RT), but conformation-selective interactions elicited by the linker. Specifically, the R linker that in the R:C complex docks in the active site of C in apo R preferentially interacts with the C-binding incompetent state of the adjacent cAMP-binding domain (CBD). These unanticipated findings imply that the formation of the intermolecular R:C inhibitory interface occurs at the expense of destabilizing the intramolecular linker/CBD interactions in R. A direct implication of this model, which was not predictable solely based on protein structure, is that the disruption of a linker/CBD salt bridge in the R:C complex unexpectedly leads to increased affinity of R for C. The linker includes therefore sites of R:C complex frustration and frustration-relieving mutations enhance the kinase inhibitory potency of R without compromising its specificity.R egulation of signaling systems often relies on multidomain proteins, which function as conformational switches controlled by allosteric effectors (1-13). The structural and dynamic changes experienced by folded domains during effector-dependent allosteric transitions have been extensively investigated (1-21). However, the current understanding of the allosteric role of partially unstructured linkers is at best scant. Although the role of covalent linkage in colocalization of protein domains is well known, it has recently been hypothesized that linkers, although generally quite flexible, have evolved to serve not simply as passive covalent threads connecting one domain to the next (i.e., "beadson-a-string" model), but also as active components of functionally relevant allosteric networks (22)(23)(24)(25). To test this hypothesis, here we have investigated the allosteric role of a critical linker in the regulatory subunit (R) of protein kinase A (PKA). This linker bridges the inhibitory site (IS) and a critical cAMP-binding domain (CBD) of R [i.e., RIα (119-244) or CBD domain A (CBD-A), ref. 26, Fig. 1A]. The R construct spanning the IS, the linker, and CBD-A [i.e., RIα (91-244) or R A in short, Fig. 1A] exhibits affinities for the catalytic subunit of PKA (C) and for cAMP as well as structures that are similar...

Template-assembled collagen-based polypeptides KTA-[Gly-(Gly-Pro-Hyp) n -NH2]3 (n = 1, 3, 5, 6; KTA is cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid, also known as the Kemp triacid) and acetyl-terminated single-chain collagen-based analogs Ac-(Gly-Pro-Hyp) n -NH2 (n = 1, 3, 5, 6, 9) were synthesized by solid phase segment condensation methods. The triple-helical propensities of these collagen analogs were investigated using circular dichroism, ultraviolet absorbance, optical rotation, and nuclear magnetic resonance measurements. The acetyl analogs, Ac-(Gly-Pro-Hyp) n -NH2 (n = 6, 9), assume a stable triple-helical conformation in H2O (0.2 mg/mL) at room temperature. By contrast, Ac-(Gly-Pro-Hyp)5-NH2 adopts a triple-helical conformation in H2O only below 18 °C at a concentration of 0.2 mg/mL. For the template-assembled collagen analogs, results show that KTA-[Gly-(Gly-Pro-Hyp) n -NH2]3 (n = 5, 6) peptides form triple-helical structures which have melting temperatures above 70 °C in H2O. These melting temperatures are much higher than those of the corresponding acetyl analogs, demonstrating the significant triple-helix-stabilizing effects of the KTA template. In addition, the KTA template facilitates triple-helical structures by dramatically accelerating triple-helix formation.

This paper reports a detailed conformational analysis by 1H NMR (DMSO-d6, 300 K) and molecular modeling of the octapeptide D-Phe1-Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7+ ++-Thr8-ol (disulfide bridged) known as sandostatin (or SMS 201-995 or octreotide) with both somatostatin-like and opioid-like bioactivities. This is the initial report on sandostatin showing that attempts to explain all NMR data using a single average conformation reveal several important inconsistencies including severe violations of mutually exclusive backbone-to-backbone NOEs. The inconsistencies are solved by assuming an equilibrium between antiparallel beta-sheet structures and conformations in which the C-terminal residues form a 3(10) helix-like fold (helical ensemble). This conformational equilibrium is consistent with previous X-ray diffraction investigations which show that sandostatin can adopt both the beta-sheet and the 3(10) helix-like secondary structure folds. In addition, indications of a conformational equilibrium between beta-sheet and helical structures are also found in solvent systems different from DMSO-d6 and for other highly bioactive analogs of sandostatin. In these cases a proper multiconformational NMR refinement is important in order to avoid conformational averaging artifacts. Finally, using the known models for somatostatin-like and opioid-like bioactivities of sandostatin analogs, the present investigation shows the potentials of the proposed structures for the design of novel sandostatin-based conformationally restricted peptidomimetics. These analogs are expected to refine the pharmacophore models for sandostatin bioactivities.

EPAC is a cAMP-dependent guanine nucleotide exchange factor that serves as a prototypical molecular switch for the regulation of essential cellular processes. Although EPAC activation by cAMP has been extensively investigated, the mechanism of EPAC autoinhibition is still not fully understood. The steric clash between the side chains of two conserved residues, L273 and F300 in EPAC1, has been previously shown to oppose the inactive-to-active conformational transition in the absence of cAMP. However, it has also been hypothesized that autoinhibition is assisted by entropic losses caused by quenching of dynamics that occurs if the inactive-to-active transition takes place in the absence of cAMP. Here, we test this hypothesis through the comparative NMR analysis of several EPAC1 mutants that target different allosteric sites of the cAMP-binding domain (CBD). Using what to our knowledge is a novel projection analysis of NMR chemical shifts to probe the effect of the mutations on the autoinhibition equilibrium of the CBD, we find that whenever the apo/active state is stabilized relative to the apo/inactive state, dynamics are consistently quenched in a conserved loop (β2-β3) and helix (α5) of the CBD. Overall, our results point to the presence of conserved and nondegenerate determinants of CBD autoinhibition that extends beyond the originally proposed L273/F300 residue pair, suggesting that complete activation necessitates the simultaneous suppression of multiple autoinhibitory mechanisms, which in turn confers added specificity for the cAMP allosteric effector.