Anne Gershenson

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.1−4 The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,5−7 and that it is often synonymous with function,8−11 disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways12 and functions as a prominent player in many regulatory processes.13−15 Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.16 They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.17,18 Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles3,19,20 and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.1,21 The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions22 and high-resolution insights into the architectures of amyloid fibrils were obtained.23,24 On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.15,25,26 One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure ​(Figure11)?27−29 Figure 1 Intracellular complexity. (A) Left: Cryo-electron tomography slice of a mammalian cell. Middle: Close-up view of cellular structures colored according to their identities: Right: Three-dimensional surface representation of the same region. Yellow, endoplasmic ...

Protein Folding in the Cell: Challenges and Progress

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, cotranslational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for in-cell stability.

Post-reductionist protein science, or putting Humpty Dumpty back together again.

In their native environments, proteins perform their biological roles in highly concentrated viscous solutions and in complex networks with numerous partners. Yet for many years, the normal practice has been to purify a protein of interest in order to characterize its structural and functional properties. In this Commentary, we discuss how protein scientists are now tackling the theoretical and methodological challenges of studying proteins in their physiological context.

Energy landscapes of functional proteins are inherently risky

Evolutionary pressure for protein function leads to unavoidable sampling of conformational states that are at risk of misfolding and aggregation. The resulting tension between functional requirements and the risk of misfolding and/or aggregation in the evolution of proteins is becoming more and more apparent. One outcome of this tension is sensitivity to mutation, in which only subtle changes in sequence that may be functionally advantageous can tip the delicate balance toward protein aggregation. Similarly, increasing the concentration of aggregation-prone species by reducing the ability to control protein levels or compromising protein folding capacity engenders increased risk of aggregation and disease. In this Perspective, we describe examples that epitomize the tension between protein functional energy landscapes and aggregation risk. Each case illustrates how the energy landscapes for the at-risk proteins are sculpted to enable them to perform their functions and how the risks of aggregation are minimized under cellular conditions using a variety of compensatory mechanisms.

Cation-π interactions as lipid-specific anchors for phosphatidylinositol-specific phospholipase C.

Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis , often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids, such as phosphatidylcholine, which is enriched in the outer leaflet of eukaryotic cells, are not well understood. A 500 ns long molecular dynamics simulation of PI-PLC at the surface of a lipid bilayer revealed a strikingly high number of interactions between tyrosines at the interfacial binding site and lipid choline groups with structures characteristic of cation-π interactions. Membrane affinities of PI-PLC tyrosine variants mostly tracked the simulation results, falling into two classes: (i) those with minor losses in affinity, Kd(mutant)/Kd(wild-type) ≤ 5 and (ii) those where the apparent Kd was 50-200 times higher than wild-type. Estimating ΔΔG for these Tyr/PC interactions from the apparent Kd values reveals that the free energy associated with class I is ~1 kcal/mol, comparable to the value predicted by the Wimley-White hydrophobicity scale. In contrast, removal of class II tyrosines has a higher energy cost: ~2.5 kcal/mol toward pure PC vesicles. These higher energies correlate well with the occupancy of the cation-π adducts throughout the MD simulation. Together, these results strongly indicate that PI-PLC interacts with PC headgroups via cation-π interactions with tyrosine residues and suggest that cation-π interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins.

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

Background: Specific interactions of peripheral membrane proteins with phosphatidylcholine (PC) are poorly characterized. Results: Staphylococcus aureus phosphatidylinositol-specific phospholipase C has poor affinity for PC. Introduction of two tyrosines generates a specific PC binding site. Conclusion: The PC choline cation interaction with amino acid π systems forms the PC-specific site. Significance: Well defined choline cation-aromatic π interactions may be a general motif to anchor proteins to PC-rich bilayers. Peripheral membrane proteins can be targeted to specific organelles or the plasma membrane by differential recognition of phospholipid headgroups. Although molecular determinants of specificity for several headgroups, including phosphatidylserine and phosphoinositides are well defined, specific recognition of the headgroup of the zwitterionic phosphatidylcholine (PC) is less well understood. In cytosolic proteins the cation-π box provides a suitable receptor for choline recognition and binding through the trimethylammonium moiety. In PC, this moiety might provide a sufficient handle to bind to peripheral proteins via a cation-π cage, where the π systems of two or more aromatic residues are within 4–5 Å of the quaternary amine. We prove this hypothesis by engineering the cation-π box into secreted phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, which lacks specific PC recognition. The N254Y/H258Y variant selectively binds PC-enriched vesicles, and x-ray crystallography reveals N254Y/H258Y binds choline and dibutyroylphosphatidylcholine within the cation-π motif. Such simple PC recognition motifs could be engineered into a wide variety of secondary structures providing a generally applicable method for specific recognition of PC.

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity INSIGHTS INTO PHOSPHATIDYLCHOLINE ACTIVATION AND SURFACE DILUTION INHIBITION

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.

Serpin latency transition at atomic resolution

Significance Inhibitory serpin plasminogen activator inhibitor 1 (PAI-1) helps regulate blood clot clearance, and high levels of active PAI-1 are associated with poor cancer prognoses and cardiovascular diseases. PAI-1 transitions from an active form to an inactive, latent form via large structural rearrangements. Our simulations provide the first realistic atomistic characterization, to our knowledge, of this transition. They correctly predict the kinetic effects of mutations and explain why PAI-1 binding to specific molecules accelerates the transition. Structures from these simulations provide templates to design drugs that speed up the latency transition, with potentially important biomedical applications. Serpins are large proteins, and the first-passage times of the simulated conformational transitions are many orders of magnitude longer than the time intervals accessible using conventional molecular dynamics. Protease inhibition by serpins requires a large conformational transition from an active, metastable state to an inactive, stable state. Similar reactions can also occur in the absence of proteases, and these latency transitions take hours, making their time scales many orders of magnitude larger than are currently accessible using conventional molecular dynamics simulations. Using a variational path sampling algorithm, we simulated the entire serpin active-to-latent transition in all-atom detail with a physically realistic force field using a standard computing cluster. These simulations provide a unifying picture explaining existing experimental data for the latency transition of the serpin plasminogen activator inhibitor-1 (PAI-1). They predict a long-lived intermediate that resembles a previously proposed, partially loop-inserted, prelatent state; correctly predict the effects of PAI-1 mutations on the kinetics; and provide a potential means to identify ligands able to accelerate the latency transition. Interestingly, although all of the simulated PAI-1 variants readily access the prelatent intermediate, this conformation is not populated in the active-to-latent transition of another serpin, α1-antitrypsin, which does not readily go latent. Thus, these simulations also help elucidate why some inhibitory serpin families are more conformationally labile than others.

Fluorescence Correlation Spectroscopy of Phosphatidylinositol-specific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes simultaneously interact with the substrate, PI, and with nonsubstrate lipids such as phosphatidylcholine (PC). For Bacillus thuringiensis PI-PLC these interactions are synergistic with maximal catalytic activity observed at low to moderate mole fractions of PC (X(PC)) and maximal binding occurring at low mole fractions of anionic lipids. It has been proposed that residues in alpha-helix B help to modulate membrane binding and that dimerization on the membrane surface both increases affinity for PC and activates PI-PLC, yielding the observed PI/PC synergy. Vesicle binding and activity measurements using a variety of PI-PLC mutants support many aspects of this model and reveal that while single mutations can disrupt anionic lipid binding and the anionic lipid/PC synergy, the residues important for PC binding are less localized. Interestingly, at high X(PC) mutations can both decrease membrane affinity and increase activity, supporting a model where reductions in wild-type activity at X(PC) > 0.6 result from both dilution of the substrate and tight membrane binding of PI-PLC, limiting enzyme hopping or scooting to the next substrate molecule. These results provide a direct analysis of vesicle binding and catalytic activity and shed light on how occupation of the activator site enhances enzymatic activity.

Single molecule enzymology: watching the reaction.

Single molecule optical microscopy can directly monitor substrate turnover by individual enzymes revealing the underlying distribution of reaction rates and enzyme conformations. These techniques are particularly useful for assessing cooperativity in multi-subunit enzymes such as beta-galactosidase, and for directly monitoring how ligand and substrate binding alter dynamic equilibria. Recent investigations of HIV reverse transcriptase have reiterated the importance of single molecule microscopy for determining how proteins move on oligonucleotides and how ligands and inhibitors affect motion. Similar investigations of membrane active enzymes allow direct imaging of protein-membrane interactions. For a large variety of systems, single molecule enzymology provides unprecedented images of how enzymes interact with their substrates and the differences between individual enzymes in a population.