Fibril treatment changes protein interactions of tau and α-synuclein in human neurons.

In several neurodegenerative disorders, the neuronal proteins tau and α-synuclein adopt aggregation-prone conformations capable of replicating within and between cells. To better understand how these conformational changes drive neuropathology, we compared the interactomes of tau and α-synuclein in the presence or the absence of recombinant fibril seeds. Human embryonic stem cells with an inducible neurogenin-2 transgene were differentiated into glutamatergic neurons expressing (1) WT 0N4R tau, (2) mutant (P301L) 0N4R tau, (3) WT α-synuclein, or (4) mutant (A53T) α-synuclein, each genetically fused to a promiscuous biotin ligase (BioID2). Neurons expressing unfused BioID2 served as controls. After treatment with fibrils or PBS, interacting proteins were labeled with biotin in situ and quantified using mass spectrometry via tandem mass tag labeling. By comparing interactions in mutant versus WT neurons and in fibril- versus PBS-treated neurons, we observed changes in protein interactions that are likely relevant to disease progression. We identified 45 shared interactors, suggesting that tau and α-synuclein function within some of the same pathways. Potential loci of shared interactions include microtubules, Wnt signaling complexes, and RNA granules. Following fibril treatment, physiological interactions decreased, whereas other interactions, including those between tau and 14-3-3 η, increased. We confirmed that 14-3-3 proteins, which are known to colocalize with protein aggregates during neurodegeneration, can promote or inhibit tau aggregation in vitro depending on the specific combination of 14-3-3 isoform and tau sequence.

Genetically Introducing Biochemically Reactive Amino Acids Dehydroalanine and Dehydrobutyrine in Proteins.

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-l-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intramolecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry.

Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximity-enhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical cross-linking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.

Palmitoylation of TEAD Transcription Factors Is Required for Their Stability and Function in Hippo Pathway Signaling.

The Hippo signaling pathway is responsible for regulating the function of TEAD family transcription factors in metazoans. TEADs, with their co-activators YAP/TAZ, are critical for controlling cell differentiation and organ size through their transcriptional activation of genes involved in cell growth and proliferation. Dysregulation of the Hippo pathway has been implicated in multiple forms of cancer. Here, we identify a novel form of regulation of TEAD family proteins. We show that human TEADs are palmitoylated at a universally conserved cysteine, and report the crystal structures of the human TEAD2 and TEAD3 YAP-binding domains in their palmitoylated forms. These structures show a palmitate bound within a highly conserved hydrophobic cavity at each protein's core. Our findings also demonstrate that this modification is required for proper TEAD folding and stability, indicating a potential new avenue for pharmacologically regulating the Hippo pathway through the modulation of TEAD palmitoylation.

Native MS Analysis of Bacteriorhodopsin and an Empty Nanodisc by Orthogonal Acceleration Time-of-Flight, Orbitrap and Ion Cyclotron Resonance.

Over the past two decades, orthogonal acceleration time-of-flight has been the de facto analyzer for solution and membrane-soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Three MS instruments are compared, the Q-ToF, Orbitrap, and the FT-ICR, to analyze, under native instrument and buffer conditions, the seven-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode), producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20-0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29-2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on all three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge states. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa), and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.

On the Analytical Superiority of 1D NMR for Fingerprinting the Higher Order Structure of Protein Therapeutics Compared to Multidimensional NMR Methods.

An important aspect in the analytical characterization of protein therapeutics is the comprehensive characterization of higher order structure (HOS). Nuclear magnetic resonance (NMR) is arguably the most sensitive method for fingerprinting HOS of a protein in solution. Traditionally, (1)H-(15)N or (1)H-(13)C correlation spectra are used as a "structural fingerprint" of HOS. Here, we demonstrate that protein fingerprint by line shape enhancement (PROFILE), a 1D (1)H NMR spectroscopy fingerprinting approach, is superior to traditional two-dimensional methods using monoclonal antibody samples and a heavily glycosylated protein therapeutic (Epoetin Alfa). PROFILE generates a high resolution structural fingerprint of a therapeutic protein in a fraction of the time required for a 2D NMR experiment. The cross-correlation analysis of PROFILE spectra allows one to distinguish contributions from HOS vs protein heterogeneity, which is difficult to accomplish by 2D NMR. We demonstrate that the major analytical limitation of two-dimensional methods is poor selectivity, which renders these approaches problematic for the purpose of fingerprinting large biological macromolecules.

Reduced amino acid specificity of mammalian tyrosyl-tRNA synthetase is associated with elevated mistranslation of Tyr codons.

Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, no significant change in cellular viability was observed. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with changes in amino acid pools, suggesting that mischarging of tRNA(Tyr) with noncognate Phe by tyrosyl-tRNA synthetase was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial tyrosyl-tRNA synthetase revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.

Energetics of intermolecular hydrogen bonds in a hydrophobic protein cavity.

This work explores the energetics of intermolecular H-bonds inside a hydrophobic protein cavity. Kinetic measurements were performed on the gaseous deprotonated ions (at the -7 charge state) of complexes of bovine ß-lactoglobulin (Lg) and three monohydroxylated analogs of palmitic acid (PA): 3-hydroxypalmitic acid (3-OHPA), 7-hydroxypalmitic acid (7-OHPA), and 16-hydroxypalmitic acid (16-OHPA). From the increase in the activation energy for the dissociation of the (Lg + X-OHPA)7⁻ ions, compared with that of the (Lg + PA)7⁻ ion, it is concluded that the ­OH groups of the X-OHPA ligands participate in strong (5-11 kcal mol⁻¹) intermolecular H-bonds in the hydrophobic cavity of Lg. The results of molecular dynamics (MD) simulations suggest that the ­OH groups of 3-OHPA and 16-OHPA act as H-bond donors and interact with backbone carbonyl oxygens, whereas the ­OH group of 7-OHPA acts as both H-bond donor and acceptor with nearby side chains. The capacity for intermolecular H-bonds within the Lg cavity, as suggested by the gas-phase measurements, does not necessarily lead to enhanced binding in aqueous solution. The association constant (Ka) measured for 7-OHPA [(2.3 ± 0.2) × 105 M⁻¹] is similar to the value for the PA [(3.8 ± 0.1) × 105 M⁻¹]; Ka for 3-OHPA [(1.1 ± 0.3) × 106 M⁻¹] is approximately three-times larger, whereas Ka for 16-OHPA [(2.3 ± 0.2) × 104 M⁻¹] is an order of magnitude smaller. Taken together, the results of this study suggest that the energetic penalty to desolvating the ligand ­OH groups, which is necessary for complex formation, is similar in magnitude to the energetic contribution of the intermolecular H-bonds.

Structural and functional analysis of the native peripherin-ROM1 complex isolated from photoreceptor cells.

Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function.

Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

Ordering of the N-terminus of human MDM2 by small molecule inhibitors.

Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel ß-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.

Deuterium kinetic isotope effects on the dissociation of a protein-fatty acid complex in the gas phase.

Deuterium kinetic isotope effects (KIEs) are reported for the first time for the dissociation of a protein-ligand complex in the gas phase. Temperature-dependent rate constants were measured for the loss of neutral ligand from the deprotonated ions of the 1:1 complex of bovine ß-lactoglobulin (Lg) and palmitic acid (PA), (Lg + PA)(n-) → Lg(n-) + PA, at the 6- and 7- charge states. At 25 °C, partial or complete deuteration of the acyl chain of PA results in a measurable inverse KIE for both charge states. The magnitude of the KIEs is temperature dependent, and Arrhenius analysis of the rate constants reveals that deuteration of PA results in a decrease in activation energy. In contrast, there is no measurable deuterium KIE for the dissociation of the (Lg + PA) complex in aqueous solution at pH 8. Deuterium KIEs were calculated using conventional transition-state theory with an assumption of a late dissociative transition state (TS), in which the ligand is free of the binding pocket. The vibrational frequencies of deuterated and non-deuterated PA in the gas phase and in various solvents (n-hexane, 1-chlorohexane, acetone, and water) were established computationally. The KIEs calculated from the corresponding differences in zero-point energies account qualitatively for the observation of an inverse KIE but do not account for the magnitude of the KIEs nor their temperature dependence. It is proposed that the dissociation of the (Lg + PA) complex in aqueous solution also proceeds through a late TS in which the acyl chain is extensively hydrated such that there is no significant differential change in the vibrational frequencies along the reaction coordinate and, consequently, no significant KIE.

Energetics of lipid binding in a hydrophobic protein cavity.

Hydrophobic bonding is central to many biochemical processes, such as protein folding and association. However, a complete description of the forces underlying hydrophobic interactions is lacking. The goal of this study was to evaluate the intrinsic energetic contributions of -CH(3), >CH(2), and -HC═CH- groups to protein-lipid binding. To this end, Arrhenius parameters were measured for dissociation of gaseous deprotonated ions (at the -7 charge state) of complexes of bovine ß-lactoglobulin (Lg), a model lipid-binding protein, and a series of saturated, unsaturated, and branched fatty acids (FA). In the gas phase, the (Lg + FA)(7-) ions adopt one of two noninterconverting structures, which we refer to as the fast and slow dissociating components. The dissociation activation energies measured for the fast components of the (Lg + FA)(7-) ions were found to correlate linearly with the association free energies measured in aqueous solution, suggesting that the specific protein-lipid interactions are preserved in the gas phase. The average contributions that the -CH(3), >CH(2), and -HC═CH- groups make to the dissociation activation energies measured for the fast components of the (Lg + FA)(7-) ions were compared with enthalpies for the transfer of hydrocarbons from the gas phase to organic solvents. For >CH(2) groups, the interior of the cavity was found to most closely resemble the relatively polar solvents acetone and N,N-dimethylformamide, which have dielectric constants (ε) of 21 and 39, respectively. For -CH(3) groups, the solvent environment most closely resembles 1-butanol (ε = 17), although the energetic contribution is dependent on the location of the methyl group in the FA. In contrast, the solvation of -HC═CH- groups is similar to that afforded by the nonpolar solvent cyclohexane (ε = 2).

Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet?

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein-ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

PADLOC: a powerful tool to assign disulfide bond connectivities in peptides and proteins by NMR spectroscopy.

The determination of the disulfide bond connectivity in a peptide or protein represents a significant challenge. It is notoriously difficult to use NMR spectroscopy to assign disulfide connectivities because NMR spectra lack direct evidence for disulfide bonds. These bonds are typically inferred from three-dimensional structure calculations, which can result in ambiguous disulfide assignment. Here, we present a new NMR based methodology, in which the disulfide connectivity is obtained by applying Bayesian rules of inference to the local topology of cysteine residues. We illustrate how this approach successfully predicts the disulfide connectivity in proteins for which crystal structures are available in the protein data bank (PDB). We also demonstrate how this methodology is used with experimental NMR data for peptides with complex disulfide topologies, including hepcidin, Kalata-B1, and µ-Conotoxin KIIIA. In the case of µ-Conotoxin KIIIA, the PADLOC connectivity (1-15,2-9,4-16) differs from previously published results; additional evidence is presented demonstrating unequivocally that this newly proposed connectivity is correct.

Fragment based drug discovery: practical implementation based on ¹9F NMR spectroscopy.

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

Evidence that water can reduce the kinetic stability of protein-hydrophobic ligand interactions.

The first quantitative comparison of the thermal dissociation rate constants measured for protein-ligand complexes in their hydrated and dehydrated states is described. Rate constants, measured using surface plasmon resonance spectroscopy, are reported for the dissociation of the 1:1 complexes of bovine ß-lactoglobulin (Lg) with the fatty acids (FA), palmitic acid (PA), and stearic acid (SA), in aqueous solution at pH 8 and at temperatures ranging from 5 to 45 °C. The rate constants are compared to values determined from time-resolved blackbody infrared radiative dissociation measurements for the gaseous deprotonated (Lg+FA)(n-) ions, where n = 6 and 7, at temperatures ranging from 25 to 66 °C. Notably, the hydrated (Lg+PA) complex is kinetically less stable than the corresponding gas phase (Lg+PA)(n-) ions at all temperatures investigated; the hydrated (Lg+SA) complex is kinetically less stable than the gaseous (Lg+SA)(n-) ions at temperatures <45 °C. The greater kinetic stability of the gaseous (Lg+FA)(n-) ions originates from significantly larger, by 11-12 kcal mol(-1), E(a) values. It is proposed that the differences in the dissociation E(a) values measured in solution and the gas phase reflect the differential hydration of the reactant and the dissociative transition state.

Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.

Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.

Resolving disulfide structural isoforms of IgG2 monoclonal antibodies by ion mobility mass spectrometry.

Recombinant monoclonal antibodies are an important class of therapeutic agents that have found widespread use for the treatment of many human diseases. Here, we have examined the utility of ion mobility mass spectrometry (IMMS) for the rapid characterization of disulfide variants in intact IgG2 monoclonal antibodies. It is shown that IMMS reveals 2 to 3 gas-phase conformer populations for IgG2s. In contrast, a single gas-phase conformer is revealed using IMMS for both an IgG1 antibody and a Cys-232 --> Ser mutant IgG2, both of which are homogeneous with respect to disulfide bonding. This provides strong evidence that the observed IgG2 gas-phase conformers are related to disulfide bond heterogeneity. Additionally, IMMS analysis of redox enriched disulfide isoforms allows assignment of the mobility peaks to established disulfide bonding patterns. These data clearly illustrate how IMMS can be used to quickly provide information on the higher order structure of antibody therapeutics.