The ␣-helix is a ubiquitous secondary structural element that is almost exclusively observed in proteins when stabilized by tertiary or quaternary interactions. However, beginning with the unexpected observations of ␣-helix formation in the isolated C-peptide in ribonuclease A, there is growing evidence that a significant percentage (0.2%) of all proteins contain isolated stable single ␣-helical domains (SAH). These SAH domains provide unique structural features essential for normal protein function. A subset of SAH domains contain a characteristic ER/K motif, composed of a repeating sequence of ϳ4 consecutive glutamic acids followed by ϳ4 consecutive basic arginine or lysine (R/K) residues. The ER/K ␣-helix, also termed the ER/K linker, has been extensively characterized in the context of the myosin family of molecular motors and is emerging as a versatile structural element for protein and cellular engineering applications. Here, we review the structure and function of SAH domains, as well as the tools to identify them in natural proteins. We conclude with a discussion of recent studies that have successfully used the modular ER/K linker for engineering chimeric myosin proteins with altered mechanical properties, as well as synthetic polypeptides that can be used to monitor and systematically modulate protein interactions within cells. Coiled-coil or Single ␣-Helical (SAH) Domain?Until recently, SAH 2 domains in natural proteins were predicted by secondary structure prediction algorithms to form a coiled-coil, in part due to the high concentration of charged and polar residues that are also the hallmark of the coiled-coil motif (1). Among the multiple folds in globular proteins that stabilize ␣-helices, the coiled-coil motif has been extensively characterized and in general is the most predictable form of tertiary protein structure (2). In the coiled-coil motif, two or more ␣-helices are individually stabilized by sequence-specific packing at consensus hydrophobic patches. Extensive studies have elicited general sequence and structure rules that govern coiled-coil interactions. Briefly, the amino acid sequence of each ␣-helix in a coiled-coil is divided into heptads (7 residues) that form nearly two complete ␣-helical turns and span 1.05 nm along the helical axis. Each amino acid in the heptad is described by its relative position, moving from the N to C terminus, using the nomenclature abcdefg. In canonical dimeric coiled-coils, the a and d positions radiate away from the core of the ␣-helix, 60°a part and offset by 0.45 nm along the helix length, and are typically occupied by aliphatic hydrophobic residues, whereas polar residues comprise the rest of the positions. As the heptad is repeated, it forms a continuous hydrophobic patch located along a single face of the ␣-helix compatible with a tight intermolecular interaction between polypeptides with the same or similar heptad pattern (2). However, often polar or charged residues occupy the a and d positions, leading to local destabilization of the coiled-coil...

We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements; however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.

Background: Protein kinase C (PKC) is a nodal regulator of cell signaling. Results: Multiple interactions between conserved regulatory domains in PKC synergistically stabilize a nanomolar affinity homodimer critical for cellular function. Conclusion: Homodimerization regulates the equilibrium between the auto-inhibited and active states of PKC␣. Significance: Multiplexed interactions between modular domains dictate PKC function.

Necroptosis, an inflammatory form of cell death, is initiated by the activation of receptor-interacting protein kinase 3 (RIPK3), which depends on its interaction with RIPK1. Although catalytically inactive, the RIPK3 mutant D161N still stimulates RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice. Whereas the absence of RIPK1 rescues RIPK3 D161N homozygous mice, we report that the absence of RIPK1 leads to embryonic lethality in RIPK3 D161N heterozygous mice. This suggested that the kinase domain of RIPK3 had a noncatalytic function that was enhanced by a conformation induced by the D161N mutation. We found that the RIPK3 kinase domain homodimerized through a surface that is structurally similar to that of the RAF family members. Mutation of residues at the dimer interface impaired dimerization and necroptosis. Kinase domain dimerization stimulated the activation of RIPK3 through cis-autophosphorylation. This noncatalytic, allosteric activity was enhanced by certain kinase-deficient mutants of RIPK3, including D161N. Furthermore, apoptosis induced by certain RIPK3 inhibitors was also dependent on the kinase dimerization interface. Our studies reveal that the RIPK3 kinase domain exhibits catalytically independent function that is important for both RIPK3-dependent necroptosis and apoptosis.

Resolving the conformational dynamics of large multidomain proteins has proven to be a significant challenge. Here we use a variety of techniques to dissect the roles of individual protein kinase Cα (PKCα) regulatory domains in maintaining catalytic autoinhibition. We find that whereas the pseudosubstrate domain is necessary for autoinhibition it is not sufficient. Instead, each regulatory domain (C1a, C1b, and C2) appears to strengthen the pseudosubstrate-catalytic domain interaction in a nucleotide-dependent manner. The pseudosubstrate and C1a domains, however, are minimally essential for maintaining the inactivated state. Furthermore, disrupting known interactions between the C1a and other regulatory domains releases the autoinhibited interaction and increases basal activity. Modulating this interaction between the catalytic and regulatory domains reveals a direct correlation between autoinhibition and membrane translocation following PKC activation.

Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKC—mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering.

Viral aggregation is a complex and pervasive phenomenon affecting many viral families. An increasing number of studies have indicated that it can modulate critical parameters surrounding viral infections, and yet its role in viral infectivity, pathogenesis, and evolution is just beginning to be appreciated. Aggregation likely promotes viral infection by increasing the cellular multiplicity of infection (MOI), which can help overcome stochastic failures of viral infection and genetic defects and subsequently modulate their fitness, virulence, and host responses. Conversely, aggregation can limit the dispersal of viral particles and hinder the early stages of establishing a successful infection. The cost–benefit of viral aggregation seems to vary not only depending on the viral species and aggregating factors but also on the spatiotemporal context of the viral life cycle. Here, we review the knowns of viral aggregation by focusing on studies with direct observations of viral aggregation and mechanistic studies of the aggregation process. Next, we chart the unknowns and discuss the biological implications of viral aggregation in their infection cycle. We conclude with a perspective on harnessing the therapeutic potential of this phenomenon and highlight several challenging questions that warrant further research for this field to advance.

Given the frequency with which MAP kinase signaling is dysregulated in cancer, much effort has been focused on inhibiting RAS signaling for therapeutic benefit. KSR1, a pseudokinase that interacts with RAF, is a potential target; it was originally cloned in screens for suppressors of constitutively active RAS, and its deletion prevents RAS-mediated transformation of mouse embryonic fibroblasts. In this work, we used a genetically engineered mouse model of pancreatic cancer to assess whether KSR1 deletion would influence tumor development in the setting of oncogenic RAS. We found that Ksr1-/- mice on this background had a modest but significant improvement in all-cause morbidity compared to Ksr1+/+ and Ksr1+/- cohorts. Ksr1-/- mice, however, still developed tumors, and precursor pancreatic intraepithelial neoplastic (PanIN) lesions were detected within a similar timeframe compared to Ksr1+/+ mice. No significant differences in pERK expression or in proliferation were noted. RNA sequencing also did not reveal any unique genetic signature in Ksr1-/- tumors. Further studies will be needed to determine whether and in what settings KSR inhibition may be clinically useful.