Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry.

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

Relative interfacial cleavage energetics of protein complexes revealed by surface collisions.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

Localization of Protein Complex Bound Ligands by Surface-Induced Dissociation High-Resolution Mass Spectrometry.

Surface-induced dissociation (SID) is a powerful means of deciphering protein complex quaternary structures due to its capability of yielding dissociation products that reflect the native structures of protein complexes in solution. Here we explore the suitability of SID to locate the ligand binding sites in protein complexes. We studied C-reactive protein (CRP) pentamer, which contains a ligand binding site within each subunit, and cholera toxin B (CTB) pentamer, which contains a ligand binding site between each adjacent subunit. SID dissects ligand-bound CRP into subcomplexes with each subunit carrying predominantly one ligand. In contrast, SID of ligand-bound CTB results in the generation of subcomplexes with a ligand distribution reflective of two subunits contributing to each ligand binding site. SID thus has potential application in localizing sites of small ligand binding for multisubunit protein-ligand complexes.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Surface-Induced Dissociation Mass Spectra as a Tool for Distinguishing Different Structural Forms of Gas-Phase Multimeric Protein Complexes.

One attractive feature of ion mobility mass spectrometry (IM-MS) lies in its ability to provide experimental collision cross section (CCS) measurements, which can be used to distinguish different conformations that a protein complex may adopt during its gas-phase unfolding. However, CCS values alone give no detailed information on subunit structure within the complex. Consequently, structural characterization typically requires molecular modeling, which can have uncertainties without experimental support. One method of obtaining direct experimental evidence on the structures of these intermediates is utilizing gas-phase activation techniques that can effectively dissociate the complexes into substructures while preserving the native topological information. The most commonly used activation method, collision-induced dissociation (CID) with low-mass target gases, typically leads to unfolding of monomers of a protein complex. Here, we describe a method that couples IM-MS and surface-induced dissociation (SID) to dissociate the source-activated precursors of three model protein complexes: C-reactive protein (CRP), transthyretin (TTR), and concanavalin A (Con A). The results of this study confirm that CID involves the unfolding of the protein complex via several intermediates. More importantly, our experiments also indicate that retention of similar CCS between different intermediates does not guarantee retention of structure. Although CID spectra (at a given collision energy) of source-activated, mass-selected precursors do not distinguish between native-like, collapsed, and expanded forms of a protein complex, dissociation patterns and/or average charge states of monomer products in SID of each of these forms are unique.

Illustration of SID-IM-SID (surface-induced dissociation-ion mobility-SID) mass spectrometry: homo and hetero model protein complexes.

The direct determination of the overall topology and inter-subunit contacts of protein complexes plays an integral role in understanding how different subunits assemble into biologically relevant multisubunit complexes. Mass spectrometry has emerged as a useful structural biological tool because of its sensitivity, high tolerance for heterogeneous mixtures and the fact that crystals are not required. Perturbation of subunit interfaces in solution followed by gas-phase detection using mass spectrometry is a current means of probing the disassembly and hence assembly of protein complexes. Herein, we present an alternative method that employs native mass spectrometry coupled with ion mobility and two stages of surface induced dissociation (SID) where protein complexes are dissociated into subcomplexes in the first SID stage. The subcomplexes are then separated by ion mobility and subsequently fragmented into their individual monomers in the second SID stage (SID-IM-SID), providing information on how individual subunits assemble into protein complexes with different native topologies. The results also illustrate complex dependent differences in charge redistribution onto individual monomers obtained in SID-IM-SID.

Surface-Induced Dissociation of Homotetramers with D2 Symmetry Yields their Assembly Pathways and Characterizes the Effect of Ligand Binding.

Understanding of protein complex assembly and the effect of ligand binding on their native topologies is integral to discerning how alterations in their architecture can affect function. Probing the disassembly pathway may offer insight into the mechanisms through which various subunits self-assemble into complexes. Here, a gas-phase dissociation method, surface-induced dissociation (SID) coupled with ion mobility (IM), was utilized to determine whether disassembly pathways are consistent with the assembly of three homotetramers and to probe the effects of ligand binding on conformational flexibility and tetramer stability. The results indicate that the smaller interface in the complex is initially cleaved upon dissociation, conserving the larger interface, and suggest that assembly of a D2 homotetramer from its constituent monomers occurs via a C2 dimer intermediate. In addition, we demonstrate that ligand-mediated changes in tetramer SID dissociation behavior are dependent on where and how the ligand binds.

A dimer interface mutation in glyceraldehyde-3-phosphate dehydrogenase regulates its binding to AU-rich RNA.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3'-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD(+) binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.

Human defensins facilitate local unfolding of thermodynamically unstable regions of bacterial protein toxins.

Defensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins' enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil ?-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric ?-defensin HD5 and ?-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.

Interfacial residues promote an optimal alignment of the catalytic center in human soluble guanylate cyclase: heterodimerization is required but not sufficient for activity.

Soluble guanylate cyclase (sGC) plays a central role in the cardiovascular system and is a drug target for the treatment of pulmonary hypertension. While the three-dimensional structure of sGC is unknown, studies suggest that binding of the regulatory domain to the catalytic domain maintains sGC in an autoinhibited basal state. The activation signal, binding of NO to heme, is thought to be transmitted via the regulatory and dimerization domains to the cyclase domain and unleashes the full catalytic potential of sGC. Consequently, isolated catalytic domains should show catalytic turnover comparable to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we show unambiguously that human isolated catalytic domains are much less active than basal sGC, while still forming heterodimers. We identified key structural elements regulating the dimer interface and propose a novel role for residues located in an interfacial flap and a hydrogen bond network as key modulators of the orientation of the catalytic subunits. We demonstrate that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. Our data support a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of heterodimerization and activation of sGC and open additional drug discovery routes for targeting the NO-sGC-cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.