Structural transitions enable interleukin-18 maturation and signaling.

Several interleukin-1 (IL-1) family members, including IL-1ß and IL-18, require processing by inflammasome-associated caspases to unleash their activities. Here, we unveil, by cryoelectron microscopy (cryo-EM), two major conformations of the complex between caspase-1 and pro-IL-18. One conformation is similar to the complex of caspase-4 and pro-IL-18, with interactions at both the active site and an exosite (closed conformation), and the other only contains interactions at the active site (open conformation). Thus, pro-IL-18 recruitment and processing by caspase-1 is less dependent on the exosite than the active site, unlike caspase-4. Structure determination by nuclear magnetic resonance uncovers a compact fold of apo pro-IL-18, which is similar to caspase-1-bound pro-IL-18 but distinct from cleaved IL-18. Binding sites for IL-18 receptor and IL-18 binding protein are only formed upon conformational changes after pro-IL-18 cleavage. These studies show how pro-IL-18 is selected as a caspase-1 substrate, and why cleavage is necessary for its inflammatory activity.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Less is more: A simple methyl-TROSY based pulse scheme offers improved sensitivity in applications to high molecular weight complexes.

The HMQC pulse sequence and variants thereof have been exploited in studies of high molecular weight protein complexes, taking advantage of the fact that fast and slow relaxing magnetization components are sequestered along two distinct magnetization transfer pathways. Despite the simplicity of the HMQC scheme an even shorter version can be designed, based on elimination of the terminal refocusing period, as a further means of increasing signal. Here we present such an experiment, and show that significant sensitivity gains, in some cases by factors of two or more, are realized in studies of proteins varying in molecular masses from 100 kDa to 1 MDa.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Interrogating the Quaternary Structure of Noncanonical Hemoglobin Complexes by Electrospray Mass Spectrometry and Collision-Induced Dissociation.

Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, "noncanonical" hexameric and octameric complexes were observed. CID of all these species [(αß)2, (αß)3, and (αß)4] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein-protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes.

Gas Phase Protein Folding Triggered by Proton Stripping Generates Inside-Out Structures: A Molecular Dynamics Simulation Study.

The properties of electrosprayed protein ions continue to be enigmatic, owing to the absence of high-resolution structure determination methods in the gas phase. There is considerable evidence that under properly optimized conditions these ions preserve solution-like conformations and interactions. However, it is unlikely that these solution-like conformers represent the "intrinsic" structural preferences of gaseous proteins. In an effort to uncover what such intrinsically preferred conformers might look like, we performed molecular dynamics (MD) simulations of gaseous ubiquitin. Our work was inspired by recent gas phase experiments, where highly extended 13+ ubiquitin ions were transformed to compact 3+ species by proton stripping (Laszlo, K. J.; Munger, E. B.; Bush, M. F. J. Am. Chem. Soc. 2016, 138, 9581-9588). Our simulations covered several microseconds and used a mobile-proton algorithm to account for the fact that a H+ in gaseous proteins can migrate between different titratable sites. Proton stripping caused folding of ubiquitin into heterogeneous "inside-out" structures. The hydrophilic core of these conformers was stabilized by charge-charge and polar interactions, while hydrophobic residues were located on the protein surface. Collision cross sections of these MD structures were in good agreement with experimental results. The inside-out structures generated during gas phase folding are in striking contrast to the solution behavior which is dominated by the hydrophobic effect, i.e., the tendency to bury hydrophobic side chains in the core (instead of exposing them to the surface). We do not dispute that native-like proteins can be transferred into the gas phase as kinetically trapped species. However, those metastable conformers do not represent the intrinsic structural preferences of gaseous proteins. Our work for the first time provides detailed insights into the properties of intrinsically preferred gas phase conformers, and we unequivocally find them to have inside-out architectures.