Melinda Roy

A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes.

The specificity of intracellular signaling events is controlled, in part, by compartmentalization of protein kinases and phosphatases. The subcellular localization of these enzymes is often maintained by protein‐ protein interactions. A prototypic example is the compartmentalization of the cAMP‐dependent protein kinase (PKA) through its association with A‐kinase anchoring proteins (AKAPs). A docking and dimerization domain (D/D) located within the first 45 residues of each regulatory (R) subunit protomer forms a high affinity binding site for its anchoring partner. We now report the structures of two D/D‐AKAP peptide complexes obtained by solution NMR methods, one with Ht31(493–515) and the other with AKAP79(392–413). We present the first direct structural data demonstrating the helical nature of the peptides. The structures reveal conserved hydrophobic interaction surfaces on the helical AKAP peptides and the PKA R subunit, which are responsible for mediating the high affinity association in the complexes. In a departure from the dimer‐dimer interactions seen in other X‐type four‐helix bundle dimeric proteins, our structures reveal a novel hydrophobic groove that accommodates one AKAP per RIIα D/D.

The molecular basis for protein kinase A anchoring revealed by solution NMR.

Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein–protein interactions. The adenosine 3´,5´-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIα(1–44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIα(1–44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein–protein interactions in signal transduction.

MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone.

Iron–sulfur (Fe–S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified outer mitochondrial membrane Fe–S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the ≈650 reported Fe–S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a β-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe–2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe–2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe–S cluster transfer.

Related Protein-Protein Interaction Modules Present Drastically Different Surface Topographies Despite A Conserved Helical Platform

The subcellular localization of cAMP-dependent protein kinase (PKA) occurs through interaction with A-Kinase Anchoring Proteins (AKAPs). AKAPs bind to the PKA regulatory subunit dimer of both type Ialpha and type IIalpha (RIalpha and RIIalpha). RIalpha and RIIalpha display characteristic localization within different cell types, which is maintained by interaction of AKAPs with the N-terminal dimerization and docking domain (D/D) of the respective regulatory subunit. Previously, we reported the solution structure of RIIa D/D module, both free and bound to AKAPs. We have now solved the solution structure of the dimerization and docking domain of the type Ialpha regulatory dimer subunit (RIalpha D/D). RIalpha D/D is a compact docking module, with unusual interchain disulfide bonds that help maintain the AKAP interaction surface. In contrast to the shallow hydrophobic groove for AKAP binding across the surface of the RIIalpha D/D dimeric interface, the RIalpha D/D module presents a deep cleft for proposed AKAP binding. RIalpha and RIIalpha D/D interaction modules present drastically differing dimeric topographies, despite a conserved X-type four-helix bundle structure.

Isoform-specific Differences between the Type Iα and IIα Cyclic AMP-dependent Protein Kinase Anchoring Domains Revealed by Solution NMR

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (1). Discovery of dual specificityanchoring proteins(d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (2). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (3). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH2 terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH2-terminal residues in RIIα contain determinants for anchoring, and the orientation and packing of this helical element in the RIα structure may have profound consequences in the recognition surface presented to the AKAPs.

Backtracking on the folding landscape of the β-trefoil protein interleukin-1β?

Interleukin-1β (IL-1β) is a cytokine within the β-trefoil family. Our data indicate that the folding/unfolding routes are geometrically frustrated. Follow-up theoretical studies predicted backtracking events that could contribute to the broad transition barrier and the experimentally observed long-lived intermediate. The backtracking route is attributed to the topological frustration introduced by the packing of the functional loop (the β-bulge, residues 47–53) to the nascent barrel. We used real-time refolding NMR experiments to test for the presence of backtracking events predicted from our theoretical studies. Structural variants of IL-1β, a β-bulge deletion, and a circular permutation that opens the protein in the middle of the experimentally observed kinetic intermediate, were also refolded and studied to determine the affects on the observed folding reactions. The functional loop deletion variant demonstrated less backtracking than in WT protein whereas the permutation still maintains backtracking in agreement with theoretical predictions. Taken together, these findings indicate that the backtracking results from geometric frustration introduced into the fold for functional purposes.

The structural basis of compstatin activity examined by structure-function-based design of peptide analogs and NMR.

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I β-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the β-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 310-helix or a β-turn. 2) The type I β-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I β-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the β-turn is essential for activity. 5) The type I β-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) β-Turn residues Gln5(Asn5)-Asp6-Trp7(Phe7)-Gly8are specific for the turn formation, but only Gln5(Asn5)-Asp6-Trp7-Gly8residues are specific for activity. 8) Trp7 is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.

Studies of Structure-Activity Relations of Complement Inhibitor Compstatin.

Compstatin, a 13-mer cyclic peptide, is a novel and promising inhibitor of the activation of the complement system. In our search for a more active analog and better understanding of structure-functions relations, we designed a phage-displayed random peptide library based on previous knowledge of structure activity relations, in which seven amino acids deemed necessary for structure and activity were kept fixed while the remaining six were optimized. Screening of this library against C3 identified four binding clones. Synthetic peptides corresponding to these clones revealed one analog, called acetylated Ile1Leu/His9Trp/Thr13Gly triple replacement analog of compstatin corresponding to clone 640 (Ac-I1L/H9W/T13G), which was more active than compstatin. This newly identified peptide had 4-fold higher activity when compared with the originally isolated form of compstatin and 1.6-fold higher activity when compared with acetylated compstatin (Ac-compstatin). The structures of Ac-I1L/H9W/T13G and Ac-compstatin were studied by nuclear magnetic resonance, compared with the structure of compstatin, and found to be very similar. The binding of Ac-I1L/H9W/T13G and the equally active acetylated analog with His9Ala replacement (Ac-H9A) to C3 was evaluated by surface plasmon resonance, which suggested similarity in their binding mechanism but difference when compared with Ac-compstatin. Compensatory effects of flexibility outside the β-turn and tryptophan ring stacking may be responsible for the measured activity increase in Ac-I1L/H9W/T13G and acetylated analog with His9Ala replacement and the variability in binding mechanism compared with Ac-compstatin. These data demonstrate that tryptophan is a key amino acid for activity. Finally, the significance of the N-terminal acetylation was examined and it was found that the hydrophobic cluster at the linked termini of compstatin is essential for binding to C3 and for activity.

Isoform-Specific Differences Between the Type Iα and IIα PKA Anchoring Domains Revealed by Solution NMR

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (1). Discovery of dual specificityanchoring proteins(d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (2). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (3). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH2 terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH2-terminal residues in RIIα contain determinants for anchoring, and the orientation and packing of this helical element in the RIα structure may have profound consequences in the recognition surface presented to the AKAPs.

β-Bulge triggers route-switching on the functional landscape of interleukin-1β

Proteins fold into three-dimensional structures in a funneled energy landscape. This landscape is also used for functional activity. Frustration in this landscape can arise from the competing evolutionary pressures of biological function and reliable folding. Thus, the ensemble of partially folded states can populate multiple routes on this journey to the native state. Although protein folding kinetics experiments have shown the presence of such routes for several proteins, there has been sparse information about the structural diversity of these routes. In addition, why a given protein populates a particular route more often than another protein of similar structure and sequence is not clear. Whereas multiple routes are observed in theoretical studies on the folding of interleukin-1β (IL-1β), experimental results indicate one dominant route where the central portion of the protein folds first, and is then followed by closure of the barrel in this β-trefoil fold. Here we show, using a combination of computation and experiment, that the presence of functionally important regions like the β-bulge in the signaling protein IL-1β strongly influences the choice of folding routes. By deleting the β-bulge, we directly observe the presence of route-switching. This route-switching provides a direct link between route selection and the folding and functional landscapes of a protein.