Michael T. Lerch

Structural basis for nucleotide exchange in heterotrimeric G proteins

How a receptor transmits a signal G protein–coupled receptors (GPCRs) transmit diverse external signals into the cell. When activated by an outside stimulus, they bind to a G protein inside the cell and accelerate exchange of a bound guanosine diphosphate (GDP) nucleotide for guanosine triphosphate, which initiates intercellular signaling. Dror et al. used atomic-level molecular dynamics simulations to show how GPCRs enhance GDP release. The G protein is dynamic and frequently adopts a conformation that exposes GDP even without the receptor bound. GPCR binding to this conformation favors an additional structural rearrangement that favors GDP release. The authors confirmed these predictions experimentally using double electron-electron resonance spectroscopy. Science, this issue p. 1361 Atomic-level simulations show how G protein–coupled receptors trigger G protein signaling. G protein–coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains—previously observed to separate widely upon receptor binding to expose the nucleotide-binding site—separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

Significance Excited states of proteins play functional roles, but their low population and conformational flexibility pose a challenge for characterization by most spectroscopic techniques. Here, this challenge is met by combining high hydrostatic pressure, which reversibly populates excited states, and site-directed spin labeling with double electron–electron resonance (DEER) spectroscopy, which resolves distinct conformational substates of proteins by measuring distances between spin-labeled pairs. We present a method for trapping high-pressure equilibria of proteins by rapid freezing under pressure, followed by depressurization and acquisition of DEER data at atmospheric pressure. The methodology is applied to myoglobin, revealing unique information on the length scale of helical fluctuations in the pressure-populated as compared with the pH-populated molten globule states of the apo-protein. The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron–electron resonance (DEER) provides long-range (20–80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0–3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Significance High hydrostatic pressure facilitates the characterization of functionally relevant, but sparsely populated, excited conformational states of proteins by reversibly increasing their equilibrium population. Here, high-pressure instrumentation for circular dichroism and developments in high-pressure site-directed spin-labeling EPR are reported, and a combination of EPR and circular dichroism is used to map pressure-populated structural changes in various states of myoglobin. The data reveal that the high-pressure molten globule (MG) of apomyoglobin at neutral pH retains native-like helical content despite a fluctuating tertiary fold, an MG state of holomyoglobin populated at low pH and high-pressure retains ligand-binding capacity, and a transient folding intermediate of apomyoglobin is populated under similar conditions at equilibrium. Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875–85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

Significance High pressure has emerged as a powerful tool for exploring the energy landscape of proteins, but structural origins of the pressure response remain controversial. The results of this study on a cavity mutant of T4 lysozyme (L99A) provide direct evidence for a structure-relaxation mechanism wherein pressure shifts conformational equilibria toward states with alternative packing arrangements that fill cavities or voids in the core. Both structure relaxation and cavity hydration can occur in response to pressure, and which dominates is found to depend on details of the energy landscape. The results also address conflicting views regarding the pressure response of L99A that have recently been published. Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306–E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron–electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111–114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2–3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.

Saturation Recovery EPR and Nitroxide Spin Labeling for Exploring Structure and Dynamics in Proteins.

Experimental techniques capable of determining the structure and dynamics of proteins are continuously being developed in order to understand protein function. Among existing methods, site-directed spin labeling in combination with saturation recovery (SR) electron paramagnetic resonance spectroscopy contributes uniquely to the determination of secondary and tertiary protein structure under physiological conditions, independent of molecular weight and complexity. In addition, SR of spin labeled proteins was recently demonstrated to be sensitive to conformational exchange events with characteristic lifetimes on the order of μs, a time domain that presents a significant challenge to other spectroscopic techniques. In this chapter, we present the theoretical background necessary to understand the capabilities of SR as applied to spin labeled proteins, the instrumental requirements, and practical experimental considerations necessary to obtain interpretable data, and the use of SR to obtain information on protein: (1) secondary structure via solvent accessibility measurements, (2) tertiary structure using interspin distance measurements, and (3) conformational exchange.

A LECTIN HPLC METHOD TO ENRICH SELECTIVELY-GLYCOSYLATED PEPTIDES FROM COMPLEX BIOLOGICAL SAMPLES

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions

SUMMARY HIV‐1 Envelope (Env) mediates viral‐host membrane fusion after binding host‐receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion‐bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand‐free, CD4‐bound, inhibitor‐bound, and antibody‐bound SOSIPs, we measured inter‐subunit distances throughout spin‐labeled SOSIPs using double electron‐electron resonance (DEER) spectroscopy and compared results to soluble and virion‐bound Env structures, and single‐molecule fluorescence resonance energy transfer (smFRET)‐derived dynamics of virion‐bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody‐bound structures and shielding of non‐neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra‐subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter‐subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion‐bound Envs and demonstrate DEERs relevance for immunogen design. Graphical Abstract Figure. No caption available. HighlightsSOSIP Env apex is 3‐fold symmetric and consistent with closed prefusion structuresUnliganded Env base and CD4‐bound Env apex and base exhibit flexibilitySOSIPs retain desired properties of immunogens; e.g., burying non‐neutralizing epitopesResults allow interpretation of smFRET studies and SOSIP and virion Env structures &NA; HIV‐1 Env, the only target of neutralizing antibodies, is highly dynamic, and only snapshots of static conformations are available. Stadtmueller et al. used DEER spectroscopy to map conformations of soluble Env and its complexes with antibodies or the CD4 receptor. Results reveal similarities to virion‐bound Env and buried non‐neutralizing antibody epitopes, advancing knowledge of Env function and vaccine design.

Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1

Significance Cellular acidification occurs in response to many cellular events, including ischemia and apoptosis. The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and has been implicated in mitochondrial regulation, cell death, and assembling protein complexes with Bcl2 family of apoptotic regulating proteins. During apoptosis, VDAC has demonstrated rapid oligomerization followed by cytochrome c release into the cytoplasm. We report a new dimeric model of VDAC induced by minor acidification with a pKa of 7.4, which is regulated by the protonation state of transmembrane residue E73. This study links a specific dimer formation with cellular acidification, which may be a salient feature of the molecular mechanism(s) involved in mitochondrial regulation. The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC’s role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron–electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer–dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress.