Nicole C. Norris

A dihydropyridine receptor α1s loop region critical for skeletal muscle contraction is intrinsically unstructured and binds to a SPRY domain of the type 1 ryanodine receptor

The II-III loop of the dihydropyridine receptor (DHPR) alpha(1s) subunit is a modulator of the ryanodine receptor (RyR1) Ca(2+) release channel in vitro and is essential for skeletal muscle contraction in vivo. Despite its importance, the structure of this loop has not been reported. We have investigated its structure using a suite of NMR techniques which revealed that the DHPR II-III loop is an intrinsically unstructured protein (IUP) and as such belongs to a burgeoning structural class of functionally important proteins. The loop does not possess a stable tertiary fold: it is highly flexible, with a strong N-terminal helix followed by nascent helical/turn elements and unstructured segments. Its residual structure is loosely globular with the N and C termini in close proximity. The unstructured nature of the II-III loop may allow it to easily modify its interaction with RyR1 following a surface action potential and thus initiate rapid Ca(2+) release and contraction. The in vitro binding partner for the II-III was investigated. The II-III loop interacts with the second of three structurally distinct SPRY domains in RyR1, whose function is unknown. This interaction occurs through two preformed N-terminal alpha-helical regions and a C-terminal hydrophobic element. The A peptide corresponding to the helical N-terminal region is a common probe of RyR function and binds to the same SPRY domain as the full II-III loop. Thus the second SPRY domain is an in vitro binding site for the II-III loop. The possible in vivo role of this region is discussed.

Structural and Functional Analysis of the Tandem β-Zipper Interaction of a Streptococcal Protein with Human Fibronectin

Bacterial fibronectin-binding proteins (FnBPs) contain a large intrinsically disordered region (IDR) that mediates adhesion of bacteria to host tissues, and invasion of host cells, through binding to fibronectin (Fn). These FnBP IDRs consist of Fn-binding repeats (FnBRs) that form a highly extended tandem β-zipper interaction on binding to the N-terminal domain of Fn. Several FnBR residues are highly conserved across bacterial species, and here we investigate their contribution to the interaction. Mutation of these residues to alanine in SfbI-5 (a disordered FnBR from the human pathogen Streptococcus pyogenes) reduced binding, but for each residue the change in free energy of binding was <2 kcal/mol. The structure of an SfbI-5 peptide in complex with the second and third F1 modules from Fn confirms that the conserved FnBR residues play equivalent functional roles across bacterial species. Thus, in SfbI-5, the binding energy for the tandem β-zipper interaction with Fn is distributed across the interface rather than concentrated in a small number of “hot spot” residues that are frequently observed in the interactions of folded proteins. We propose that this might be a common feature of the interactions of IDRs and is likely to pose a challenge for the development of small molecule inhibitors of FnBP-mediated adhesion to and invasion of host cells.

Structural and functional characterization of interactions between the dihydropyridine receptor II-III loop and the ryanodine receptor.

1 Excitation–contraction coupling in skeletal muscle is dependent on a physical interaction between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR). 2 A number of peptides derived from the II–III loop region of the DHPR have been shown to be functionally active in stimulating the release of calcium via RyR channels. Their function has been found to correlate with the presence of a basic helical region located at the N‐terminus of the II–III loop. 3 The entire recombinant skeletal DHPR II–III loop is an efficient activator of RyR1 and RyR2. 4 The skeletal DHPR II–III loop is comprised of a series of a‐helices, but its tertiary structure has been determined to be unstructured and flexible. 5 Fluorescence quenching experiments have been used to identify and measure the binding affinity of the II–III loop with fragments of the RyR.

Chromatinized Protein Kinase C-θ: Can It Escape the Clutches of NF-κB?

We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.

Molecular recognition of the disordered dihydropyridine receptor II-III loop by a conserved spry domain of the type 1 ryanodine receptor.

1 The dihydropyridine receptor (DHPR) II–III loop is an intrinsically unstructured region made up of α‐helical and β‐turn secondary structure elements with the N and C termini in close spatial proximity. 2 The DHPR II–III loop interacts in vitro with a ryanodine receptor (RyR) 1 SPRY domain through α‐helical segments located in the A and B regions. Mutations within the A and B regions in the DHPR II–III loop alter the binding affinity to the SPRY2 domain. 3 The A and C peptides derived from DHPR II–III loop show negative cooperativity in binding to the SPRY2 domain. 4 The SPRY2 domain of the RyR1 (1085–1208) forms a β‐sheet sandwich structure flanked by variable loop regions. An acidic loop region of SPRY2 (1107–1121) forms part of a negatively charged cleft that is implicated in the binding of the DHPR II–III loop. 5 The mutant E1108A located in the negatively charged loop of SPRY2 reduces the binding affinity to the DHPR II–III loop.

Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β1a. Removal of the intrinsically disordered N and C termini and the hook region of β1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2–3 °C between the β1a and β2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6–8 °C. Similar to other β isoforms, β1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β1a while also providing a conduit for allosteric signaling events.