Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)-and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29 (3):606-618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1-Bcl-2 complex. NAF-1 binds to both the pro-and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein-protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1-Bcl-2 complex in the regulation of apoptosis/ autophagy in cancer biology.ife requires a controlled balance of energy conversion and utilization. These critical processes are governed by an elaborate set of reactions involving numerous protein-protein interactions. Among them is the ability of organisms to control the recycling of high-energy compounds and to control cell proliferation. These processes are, at least in part, under the control of cell survival and programmed cell death (autophagic and apoptotic) processes. Misregulation of these processes leads to many diseases, including cancer. Among the key proteins involved in these processes are Bcl-2 (1, 2) and the more recently identified iron-sulfur (Fe-S) protein nutrient-deprivation autophagy factor-1 (NAF-1) (also known as Cisd2, Miner1, Eris, and Noxp70) (3-5).NAF-1 is important for human health and disease. Missplicing of NAF-1 causes Wolfram syndrome 2 (6). NAF-1 is also functionally linked to the regulation of autophagy in cancer, and aging (3-5, 7, 8). This protein is a member of the 2Fe-2S cluster NEET family. NAF-1 has a similar backbone fold and 3Cys-1His coordination of the 2Fe-2S cluster as found in the founding member of the NEET family, mitoNEET (mNT). NAF-1 differs from mNT in the distribution of charged and aromatic surface residues (9, 10). These differences alter the 3D shape and electrostatics of the surfaces of mNT and NAF-1, leading to interactions with distinct binding partners. In fact, recent work identifi...

SR proteins are essential splicing factors whose phosphorylation by the SRPK family of protein kinases regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, non-homologous spacer insert domain that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, hydrogen-deuterium exchange and steady-state kinetic experiments were performed on SRPK1. Unlike the kinase core, the large spacer insert domain lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and spacer insert domain equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to stable structure to facilitate high affinity SR protein interactions and phosphorylation rates.

Although phosphorylation directs serine-arginine (SR) proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study we investigated the interaction of protein phosphatase 1 (PP1) with the SR protein SRSF1 to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. Disruption of RRM1-PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, re-directing SRSF1 in the nucleus. The data imply that an allosteric SR protein-phosphatase platform balances phosphorylation levels in a “goldilocks” region for the proper subnuclear storage of an SR protein splicing factor.

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce “off pathway” targeting. We now provide evidence that the nuclear protein kinase CLK1 (Cdc2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates, the SR (arginine-serine-rich) protein family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 compared to the general substrate myelin basic protein. In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in subnuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against nonphysiological targets.

Interleukin-1␤ (IL-1␤) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314 -1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1␤ or pro-IL-1␤). Pro-IL-1␤ requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1␤ and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1␤ in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1␤, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1␤/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1␤ is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.Nearly all cell types respond to interleukin (IL)-1␤, 4 in a very sensitive manner, via binding to the interleukin-1 receptor type 1 (IL-1RI) (2). Although essential in the immune response, overproduction of IL-1␤ can lead to both acute (sepsis) as well as chronic (rheumatoid arthritis, atherosclerosis, obesity, and diabetes) disease states (3). Thus, the expression, activation, and secretion of this cytokine are tightly controlled (4). Although many cell types express IL-1␤, it is predominately produced and secreted by monocytes and macrophages (1). The protein is synthesized as a biologically inactive 269-residue precursor molecule, pro-interleukin-1␤ (pro-IL-1␤), and the 153-residue active mature IL-1␤ is generated from the C-terminal domain. Processing of the proprotein involves the recently discovered NALP-1 and NALP-3 inflammasomes, which are responsible for activating procaspase-1 (5). The inflammasome function is integral in wound repair as well as for combating infection (6 -9).In vivo, the 31-kDa pro-IL-1␤ precursor is processed to the active C-terminal 17-kDa form by the interleukin-1 converting enzyme, caspase-1 (10, 11). Caspase-1 is a cysteine protease that recognizes two cleavage sites in pro-IL-1␤, the Asp 27 2Gly 28 and Asp 116 2Ala 117 peptide bonds (Fig. 1A). These cleavage sites are conserved across mammals (12-14). The activation pathway is believed to proceed with cleavage first at Asp 27 2Gly 28 (site 1) followed by Asp 116 2Ala 117 (site 2). These processing events lead ...