Helena Hernández

Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry.

The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.

Subunit architecture of multimeric complexes isolated directly from cells

Recent developments in purification strategies, together with mass spectrometry (MS)‐based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap‐binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three‐dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Recognition of a signal peptide by the signal recognition particle

Targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. Secretory and membrane proteins usually contain an amino-terminal signal peptide, which is recognized by the signal recognition particle (SRP) when nascent polypeptide chains emerge from the ribosome. The SRP–ribosome nascent chain complex is then targeted through its GTP-dependent interaction with SRP receptor to the protein-conducting channel on endoplasmic reticulum membrane in eukaryotes or plasma membrane in bacteria. A universally conserved component of SRP (refs 1, 2), SRP54 or its bacterial homologue, fifty-four homologue (Ffh), binds the signal peptides, which have a highly divergent sequence divisible into a positively charged n-region, an h-region commonly containing 8–20 hydrophobic residues and a polar c-region. No structure has been reported that exemplifies SRP54 binding of any signal sequence. Here we have produced a fusion protein between Sulfolobus solfataricus SRP54 (Ffh) and a signal peptide connected via a flexible linker. This fusion protein oligomerizes in solution through interaction between the SRP54 and signal peptide moieties belonging to different chains, and it is functional, as demonstrated by its ability to bind SRP RNA and SRP receptor FtsY. We present the crystal structure at 3.5 Å resolution of an SRP54–signal peptide complex in the dimer, which reveals how a signal sequence is recognized by SRP54.

Subunit architecture of intact protein complexes from mass spectrometry and homology modeling.

Proteomic studies have yielded detailed lists of protein components. Relatively little is known, however, of interactions between proteins or of their spatial arrangement. To bridge this gap, we are developing a mass spectrometry approach based on intact protein complexes. By studying intact complexes, we show that we are able to not only determine the stoichiometry of all subunits present but also deduce interaction maps and topological arrangements of subunits. To construct an interaction network, we use tandem mass spectrometry to define peripheral subunits and partial denaturation in solution to generate series of subcomplexes. These subcomplexes are subsequently assigned using tandem mass spectrometry. To facilitate this assignment process, we have developed an iterative search algorithm (SUMMIT) to both assign protein subcomplexes and generate protein interaction networks. This software package not only allows us to construct the subunit architecture of protein assemblies but also allows us to explore the limitations and potential of our approach. Using series of hypothetical complexes, generated at random from protein assemblies containing between six and fourteen subunits, we highlight the significance of tandem mass spectrometry for defining subunits present. We also demonstrate the importance of pairwise interactions and the optimal numbers of subcomplexes required to assign networks with up to fourteen subunits. To illustrate application of our approach, we describe the overall architecture of two endogenous protein assemblies isolated from yeast at natural expression levels, the 19S proteasome lid and the RNA exosome. In constructing our models, we did not consider previous electron microscopy images but rather deduced the subunit architecture from series of subcomplexes and our network algorithm. The results show that the proteasome lid complex consists of a bicluster with two tetrameric lobes. The exosome lid, by contrast, is a six-membered ring with three additional bridging subunits that confer stability to the ring and with a large subunit located at the base. Significantly, by combining data from MS and homology modeling, we were able to construct an atomic model of the yeast exosome. In summary, the architectural and atomic models of both protein complexes described here have been produced in advance of high-resolution structural data and as such provide an initial model for testing hypotheses and planning future experiments. In the case of the yeast exosome, the atomic model is validated by comparison with the atomic structure from X-ray diffraction of crystals of the reconstituted human exosome, which is homologous to that of the yeast. Overall therefore this mass spectrometry and homology modeling approach has given significant insight into the structure of two previously intractable protein complexes and as such has broad application in structural biology.

Protein Complexes Are under Evolutionary Selection to Assemble via Ordered Pathways

Summary Is the order in which proteins assemble into complexes important for biological function? Here, we seek to address this by searching for evidence of evolutionary selection for ordered protein complex assembly. First, we experimentally characterize the assembly pathways of several heteromeric complexes and show that they can be simply predicted from their three-dimensional structures. Then, by mapping gene fusion events identified from fully sequenced genomes onto protein complex assembly pathways, we demonstrate evolutionary selection for conservation of assembly order. Furthermore, using structural and high-throughput interaction data, we show that fusion tends to optimize assembly by simplifying protein complex topologies. Finally, we observe protein structural constraints on the gene order of fusion that impact the potential for fusion to affect assembly. Together, these results reveal the intimate relationships among protein assembly, quaternary structure, and evolution and demonstrate on a genome-wide scale the biological importance of ordered assembly pathways.

A mass spectrometry-based hybrid method for structural modeling of protein complexes

We describe a method that integrates data derived from different mass spectrometry (MS)-based techniques with a modeling strategy for structural characterization of protein assemblies. We encoded structural data derived from native MS, bottom-up proteomics, ion mobility–MS and chemical cross-linking MS into modeling restraints to compute the most likely structure of a protein assembly. We used the method to generate near-native models for three known structures and characterized an assembly intermediate of the proteasomal base.

The Interaction of the Molecular Chaperone α-Crystallin with Unfolding α-Lactalbumin: A Structural and Kinetic Spectroscopic Study

The unfolding of the apo and holo forms of bovine α-lactalbumin (α-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein α-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and 1H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with α-crystallin, have been obtained. α-LA contains four disulphide bonds and binds a calcium ion. In apo α-LA, the disulphide bonds are reduced completely over a period of ∼1500 seconds. Fully reduced α-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of α-crystallin, this precipitation can be prevented via complexation with the chaperone. α-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo α-LA but does stabilise the molten globule state. In holo α-LA, the disulphide bonds are less accessible to DTT, because of the stabilisation of the protein by the bound calcium ion, and reduction occurs much more slowly. A two-disulphide intermediate aggregates and precipitates rapidly. Its precipitation can be prevented only in the presence of a 12-fold mass excess of α-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of α-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of α-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo α-LA following dilution from denaturant are not affected by the presence of α-crystallin. Thus, α-crystallin is not a chaperone that is involved in protein folding per se. Rather, its role is to stabilise compromised, partly folded, molten globule states of proteins that are destined for precipitation.

Principles of assembly reveal a periodic table of protein complexes.

The principles of protein assembly A knowledge of protein structure greatly enhances our understanding of protein function. In many cases, function depends on oligomerization. Ahnert et al. used mass spectrometry data together with a large-scale analysis of structures of protein complexes to examine the fundamental steps of protein assembly. Systematically combining assembly steps revealed a large set of quaternary topologies that were organized into a periodic table. Based on this table, the authors accurately predicted the expected frequencies of quaternary structure topologies. Science, this issue p. 10.1126/science.aaa2245 Understanding the organizing principles allows exhaustive enumeration of possible protein quaternary structure topologies. INTRODUCTION The assembly of proteins into complexes is crucial for most biological processes. The three-dimensional structures of many thousands of homomeric and heteromeric protein complexes have now been determined, and this has had a broad impact on our understanding of biological function and evolution. Despite this, the organizing principles that underlie the great diversity of protein quaternary structures observed in nature remain poorly understood, particularly in comparison with protein folds, which have been extensively classified in terms of their architecture and evolutionary relationships. RATIONALE In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization. Our approach was to consider protein complexes in terms of their assembly. Many protein complexes assemble spontaneously via ordered pathways in vitro, and these pathways have a strong tendency to be evolutionarily conserved. Furthermore, there are strong similarities between protein complex assembly and evolutionary pathways, with assembly pathways often being reflective of evolutionary histories, and vice versa. This suggests that it may be useful to consider the types of protein complexes that have evolved from the perspective of what assembly pathways are possible. RESULTS We first examined the fundamental steps by which protein complexes can assemble, using electrospray mass spectrometry experiments, literature-curated assembly data, and a large-scale analysis of protein complex structures. We found that most assembly steps can be classified into three basic types: dimerization, cyclization, and heteromeric subunit addition. By systematically combining different assembly steps in different ways, we were able to enumerate a large set of possible quaternary structure topologies, or patterns of key interfaces between the proteins within a complex. The vast majority of real protein complex structures lie within these topologies. This enables a natural organization of protein complexes into a “periodic table,” because each heteromer can be related to a simpler symmetric homomer topology. Exceptions are mostly the result of quaternary structure assignment errors, or cases where sequence-identical subunits can have different interactions and thus introduce asymmetry. Many of these asymmetric complexes fit the paradigm of a periodic table when their assembly role is considered. Finally, we implemented a model based on the periodic table, which predicts the expected frequencies of each quaternary structure topology, including those not yet observed. Our model correctly predicts quaternary structure topologies of recent crystal and electron microscopy structures that are not included in our original data set. CONCLUSION This work explains much of the observed distribution of known protein complexes in quaternary structure space and provides a framework for understanding their evolution. In addition, it can contribute considerably to the prediction and modeling of quaternary structures by specifying which topologies are most likely to be adopted by a complex with a given stoichiometry, potentially providing constraints for multi-subunit docking and hybrid methods. Lastly, it could help in the bioengineering of protein complexes by identifying which topologies are most likely to be stable, and thus which types of essential interfaces need to be engineered. Protein assembly steps lead to a periodic table of protein complexes and can predict likely quaternary structure topologies. Three main assembly steps are possible: cyclization, dimerization, and subunit addition. By combining these in different ways, a large set of possible quaternary structure topologies can be generated. These can be arranged on a periodic table that describes most known complexes and that can predict previously unobserved topologies. Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

The flight of macromolecular complexes in a mass spectrometer

The discovery that conditions can be found such that non–covalent macromolecular complexes can survive the transition from solution to gas phase and remain intact during their flight in a mass spectrometer is an intriguing observation. While the nature of the interaction between the components, either ionic, hydrophobic or van der Waals, undoubtedly has an effect on the stability of these gas phase species, the role of small molecules in conferring additional stability is often overlooked. Here we review historical aspects of the development of mass spectrometry for macromolecular complexes with particular focus on the role of small molecules in stabilizing gas–phase complexes. Moreover, we demonstrate how the dissociation of small molecules from subunits within a macromolecular complex can be used to probe the topological arrangement. Overall, therefore, we show that mass spectrometry used in this way is capable of addressing features of the energy landscape not readily accessed by traditional structural biology approaches.

The role of salt bridges, charge density, and subunit flexibility in determining disassembly routes of protein complexes.

Summary Mass spectrometry can be used to characterize multiprotein complexes, defining their subunit stoichiometry and composition following solution disruption and collision-induced dissociation (CID). While CID of protein complexes in the gas phase typically results in the dissociation of unfolded subunits, a second atypical route is possible wherein compact subunits or subcomplexes are ejected without unfolding. Because tertiary structure and subunit interactions may be retained, this is the preferred route for structural investigations. How can we influence which pathway is adopted? By studying properties of a series of homomeric and heteromeric protein complexes and varying their overall charge in solution, we found that low subunit flexibility, higher charge densities, fewer salt bridges, and smaller interfaces are likely to be involved in promoting dissociation routes without unfolding. Manipulating the charge on a protein complex therefore enables us to direct dissociation through structurally informative pathways that mimic those followed in solution.