Human mitochondrial AAA+ ATPase SKD3/CLPB assembles into nucleotide-stabilized dodecamers.

SKD3, also known as human CLPB, belongs to the AAA+ family of ATPases associated with various activities. Mutations in the SKD3/CLPB gene cause 3-methylglutaconic aciduria type VII and congenital neutropenia. SKD3 is upregulated in acute myeloid leukemia, where it contributes to anti-cancer drug resistance. SKD3 resides in the mitochondrial intermembrane space, where it forms ATP-dependent high-molecular weight complexes, but its biological function and mechanistic links to the clinical phenotypes are currently unknown. Using sedimentation equilibrium and dynamic light scattering, we show that SKD3 is monomeric at low protein concentration in the absence of nucleotides, but it forms oligomers at higher protein concentration or in the presence of adenine nucleotides. The apparent molecular weight of the nucleotide-bound SKD3 is consistent with self-association of 12 monomers. Image-class analysis and averaging from negative-stain electron microscopy (EM) of SKD3 in the ATP-bound state visualized cylinder-shaped particles with an open central channel along the cylinder axis. The dimensions of the EM-visualized particle suggest that the SKD3 dodecamer is formed by association of two hexameric rings. While hexameric structure has been often observed among AAA+ ATPases, a double-hexamer sandwich found for SKD3 appears uncommon within this protein family. A functional significance of the non-canonical structure of SKD3 remains to be determined.

Cancer-Associated Mutations Perturb the Disordered Ensemble and Interactions of the Intrinsically Disordered p53 Transactivation Domain.

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein-protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.

Expression and Characterization of Manduca sexta Stress Responsive Peptide-1; an Inducer of Antimicrobial Peptide Synthesis.

In response to stress conditions such as wounding or infections in insects, several short peptides are processed to act as cytokines that induce AMP gene expression. To study their structure-activity relationship, immune inducibility, tissue specificity, stress responsiveness, and development relatedness, we chemically synthesized Manduca sexta stress response peptide-1, a 25-residue peptide with one disulfide bond (SRP1: FGVRVGTCPSGYVRRGTFCFPDDDY). Upon injection of the SRP1 into naïve larvae, several antimicrobial peptide genes were expressed at higher levels. The mRNA levels of SRP1 increased significantly in hemocytes and fat body after larvae were challenged with a mixture of bacteria and ß-1,3-glucan. The expression patterns of SRP1 and its target genes are somewhat different from SRP2's, suggesting overlapping yet distinct functions. We elucidated the 3D structure of SRP1 in solution by two-dimensional 1H-1H NMR spectroscopy. The tertiary structure of SRP1 consists of two short ß-strands at Y12-R15 and F18-F20, one type-II ß-turn at R15-F18 in its well-defined core and is stabilized by a covalent disulfide bond between C8 and C19. The conformational ensemble of SRP1 from extensive atomistic simulation in explicit solvent (with 3.0 µs total effective sampling) shows high consistency with experimental intramolecular NOEs of the core region. The SRP1 core adopts a fold similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF), suggesting that SRP1 may interact with EGF receptor-like molecules to trigger its biological function.

Solution Structure and Expression Profile of an Insect Cytokine: Manduca sexta Stress Response Peptide-2.

Manduca sexta stress response peptide-2 (SRP2) is predicted to be a 25-residue peptide (FGVKDGKCPSGRVRRLGICVPDDDY), which may function as an insect cytokine to regulate immune responses. Produced as an inactive precursor, endogenous proSRP2 is probably converted to active SRP2 by limited proteolysis in response to invading pathogens, along with prophenoloxidase and pro-Spätzle activation. In addition to immunity, SRP2 may control head morphogenesis or other developmental processes in the lepidopteran insect. We have examined the profiles of SRP2 gene expression in terms of immune induction capacity, tissue specificity, and developmental changes. To gain insights into its functions, we chemically synthesized SRP2, injected the peptide solution into naïve larvae, and detected significant up-regulation of several antimicrobial peptide genes. We determined the 3D molecular structure in solution of SRP2 by two-dimensional 1H-1H NMR spectroscopy. SRP2 has an ordered structure, which is composed of two short ß-strands at regions R12 - R15 and I18 - V20, one type-I' ß-turn at region R15 - I18, and a half turn at region C8 - S10 in its welldefined core stabilized by a covalent disulfide bond between C8 and C19. The secondary and tertiary structures are further stabilized by hydrogen bonds. Possible relationships between the structure and function are also discussed.