Buried Ionizable Residues Research Articles

Electrostatic Modulation of Intramolecular and Intermolecular Interactions during the Formation of an Amyloid-like Assembly.

The mechanism of protein aggregation can be broadly viewed as a shift from the native-state stabilizing intramolecular to the aggregated-phase sustaining intermolecular interactions. Understanding the role of electrostatic forces on the extent of modulation of this switch has recently evolved as a topic of monumental significance as protein aggregation has lately been connected to charge modifications of an aging proteome. To decipher the distinctive role of electrostatic forces on the extremely complicated phase separation landscape, we opted for a combined in vitro-in silico approach to ascertain the structure-dynamics-stability-aggregability relationship of the functional tandem RRM domains of the ALS-related protein TDP-43 (TDP-43tRRM), under a bivariate solution condition in terms of pH and salt concentration. Under acidic pH conditions, the native TDP-43tRRM protein creates an aggregation-prone entropically favorable partially unfolded conformational landscape due to enthalpic destabilization caused by the protonation of the buried ionizable residues and consequent overwhelming fluctuations of selective segments of the sequence leading to anti-correlated movements of the two domains of the protein. The evolved fluffy ensemble with a comparatively exposed backbone then easily interacts with incoming protein molecules in the presence of salt via typical amyloid-aggregate-like intermolecular backbone hydrogen bonds with a considerable contribution originating from the dispersion forces. Subsequent exposure to excess salt at low pH conditions expedites the aggregation process via an electrostatic screening mechanism where salt shows preferential binding to the positively charged side chain. The applied target observable-specific approach complementarity unveils the hidden information landscape of an otherwise complex process with unquestionable conviction.

Protonation-Deprotonation Switch Controls the Amyloid-like Misfolding of Nucleic-Acid-Binding Domains of TDP-43.

Nutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43tRRM) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43tRRM forms a misfolded amyloid-like protein assembly, β form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-β transition. We observed that D105A and H256Q resembled TDP-43tRRM in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the β form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43tRRM and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

M2 amphipathic helices facilitate pH-dependent conformational transition in influenza A virus

The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite λ-dynamics approach (CpHMDMSλD) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMDMSλD provides a realistic pH-dependent model for membrane proteins.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich "β form" in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

Experimental Strategy for Engineering pH Switch Proteins

Dysregulation of pH is one of the hallmarks of solid cancerous tumors. An increase in extrusion of acid from tumor cells leads to acidification of the extracellular environment, which in tumors is 0.4 to 0.8 pH units lower than in normal cells. The efficiency and selectivity of proteins (e.g. toxins and antibodies) designed to identify and kill tumor cells would be enhanced if they could be engineered to function as pH switches that are activated/deactivated by small changes in pH in the physiological range. We have demonstrated that buried ionizable residues (Lys, Glu and Asp) are useful for this purpose. Most buried ionizable residues titrate with anomalous pKa values (depressed for Lys and elevated for Glu/Asp) simply by virtue of being buried. This ensures that the residue is neutral when buried in the hydrophobic interior of the protein, consistent with expectations based on the energetic cost of removing a charged moiety from water. The shifts in pKa increase the pH sensitivity of thermodynamic stability. Using staphylococcal nuclease as a test system we engineered many variants with pairs of ionizable groups (Lys or Glu with anomalous pKa values) and demonstrated that this was sufficient to convert this protein into a pH switch that unfolds cooperatively near pH 7. Constructs with pairs of buried Lys residues are folded at high pH and unfolded by decreasing pH and constructs with pairs of buried Glu or Asp are folded at low pH and unfolded by increasing pH. Systematic engineering of several switch proteins with ionizable groups buried in different locations demonstrated that this approach is robust. Based on general thermodynamic principles, this strategy should be transferable to other proteins.

Reduction potential calculations of the Fe-S clusters in Thermus thermophilus respiratory complex I.

Respiratory complex I facilitates electron transfer from NADH to quinone over ~95 Å through a chain of seven iron-sulfur (Fe-S) clusters in the respiratory chain. In this study, the reduction potentials of the Fe-S clusters in Thermus thermophilus complex I are calculated using a Density Functional Theory + Poisson-Boltzmann method. Our results indicate that the reduction potentials are influenced by a variety of factors including the clusters being deeply buried in the complex and the protonation state of buried ionizable residues. In addition, as several of the ionizable side chains have predicted pKa values near pH 7, relatively small structural fluctuations could lead to significant (0.2 V) shifts in the reduction potential of several of the Fe-S clusters, suggesting a dynamic mechanism for electron transfer. Moreover, the method used here is a useful computational tool to study other questions about complex I. © 2019 Wiley Periodicals, Inc.

Modification of the Sweetness and Stability of Sweet-Tasting Protein Monellin by Gene Mutation and Protein Engineering.

Natural sweet protein monellin has a high sweetness and low calorie, suggesting its potential in food applications. However, due to its low heat and acid resistance, the application of monellin is limited. In this study, we show that the thermostability of monellin can be improved with no sweetness decrease by means of sequence, structure analysis, and site-directed mutagenesis. We analyzed residues located in the α-helix as well as an ionizable residue C41. Of the mutants investigated, the effects of E23A and C41A mutants were most remarkable. The former displayed significantly improved thermal stability, while its sweetness was not changed. The mutated protein was stable after 30 min incubation at 85°C. The latter showed increased sweetness and slight improvement of thermostability. Furthermore, we found that most mutants enhancing the thermostability of the protein were distributed at the two ends of α-helix. Molecular biophysics analysis revealed that the state of buried ionizable residues may account for the modulated properties of mutated proteins. Our results prove that the properties of sweet protein monellin can be modified by means of bioinformatics analysis, gene manipulation, and protein modification, highlighting the possibility of designing novel effective sweet proteins based on structure-function relationships.

Carboxyl-peptide plane stacking is important for stabilization of buried E305 of Trichoderma reesei Cel5A.

Hydrogen bonds or salt bridges are usually formed to stabilize the buried ionizable residues. However, such interactions do not exist for two buried residues D271 and E305 of Trichoderma reesei Cel5A, an endoglucanase. Mutating D271 to alanine or leucine improves the enzyme thermostability quantified by the temperature T50 due to the elimination of the desolvation penalty of the aspartic acid. However, the same mutations for E305 decrease the enzyme thermostability. Free energy calculations based on the molecular dynamics simulation predict the thermostability of D271A, D271L, and E305A (compared to WT) in line with the experimental observation but overestimate the thermostability of E305L. Quantum mechanical calculations suggest that the carboxyl-peptide plane stacking interactions occurring to E305 but not D271 are important for the carboxyl group stabilization. For the protonated carboxyl group, the interaction energy can be as much as about -4 kcal/mol for parallel stacking and about -7 kcal/mol for T-shaped stacking. For the deprotonated carboxyl group, the largest interaction energies for parallel stacking and T-shaped stacking are comparable, about -7 kcal/mol. The solvation effect generally weakens the interaction, especially for the charged system. A search of the carboxyl-peptide plane stacking in the PDB databank indicates that parallel stacking but not T-shaped stacking is quite common, and the most probable distance between the two stacking fragments is close to the value predicted by the QM calculations. This work highlights the potential role of carboxyl amide π-π stacking in the stabilization of aspartic acid and glutamic acid in proteins.

Uncovering pH-dependent transient states of proteins with buried ionizable residues.

The role of pH in regulating biological activity is ubiquitous, and understanding pH-mediated activity has traditionally relied on analyzing static biomolecular structures of highly populated ground states solved near physiological pH. However, recent advances have shown the increasing importance of transiently populated states, the characterization of which is extremely challenging but made plausible with the development of techniques such as relaxation dispersion NMR spectroscopy. To unlock the pH dependence of these transient states with atomistic-level details, we applied the recently developed explicit solvent constant pH molecular dynamics (CPHMDMSλD) framework to a series of staphylococcal nuclease (SNase) mutants with buried ionizable residues and probed their dynamics in different pH environments. Among our key findings is the existence of open states in all SNase mutants containing “buried” residues with highly shifted pKa’s, where local solvation around the protonation site was observed. The calculated pKa demonstrated good agreement with experimental pKa’s, with a low average unsigned error of 1.3 pKa units and correlation coefficient R2 = 0.78. Sampling both open and closed states in their respective pH range, where they are expected to be dominant, was necessary to reproduce experimental pKa’s, and in the most extreme examples of pKa shifts measured, it can be interpreted that the open-state structures are transient at physiological pH, contributing a small population of 1–2%. This suggests that buried ionizable residues can trigger conformational fluctuations that may be observed as transient-state structures at physiological pH. Furthermore, the coupled relationship of both open and closed states and their role in recapitulating macroscopic experimental observables suggest that structural analysis of buried residues may benefit from looking at structural pairs, as opposed to the conventional approach of looking at a single static ground-state conformation.

Identification and Characterization of Buried Ionizable Groups in Proteins

Ionizable groups buried in the hydrophobic interior of proteins play many essential biological roles. their physical properties are not well understood. Owing to the inherent differences in the dielectric properties of proteins and of water the properties of internal and surface ionizable groups can be very different. In a previous systematic study from this laboratory Asp, Glu, Lys and Arg residues were buried at 25 internal locations in staphylococcal nuclease. Most of these internal residues titrate with highly anomalous pKa values. Many crystal structures of these variants show that the majority of these buried ionizable residues are buried completely and in very diverse microenvironments. A survey of the properties and microenvironments of naturally occurring internal ionizable groups in proteins was undertaken with a large set of proteins from the Protein Data Bank to compare against the internal ionizable groups buried artificially in nuclease. Internal ionizable groups were identified based on accessible surface area and depth of burial using a self-consistent definition of the interface between protein and bulk water. For purposes of this survey, internal ionizable groups were characterized primarily in terms of accessible surface area, depth of burial, polarity and hydrophobicity of their microenvironments, and contact with other surface or internal ionizable groups and with crystallographic water molecules. MD simulations were also performed on a small set of proteins to examine the robustness of static structures for purposes of determination of depth of burial.

The High Dielectric Constant of Staphylococcal Nuclease Is Encoded in Its Structural Architecture

The pK(a) values of Lys-66, Glu-66, and Asp-66 buried in the interior of the staphylococcal nuclease Δ+PHS variant were reported to be shifted by as much as 5 pK(a) units from their normal values. Reproducing the pK(a) of these buried ionizable residues using continuum electrostatic calculations required the use of a high protein dielectric constant of 10 or higher. The apparent high dielectric constant has been rationalized as a consequence of a local structural reorganization or increased fluctuations in the microenvironment of the mutation site (Chimenti, M. S., et al. J. Mol. Biol. 2011, 405, 361-377). We have calculated the dielectric constant of Δ+PHS and the Lys-66, Asp-66, and Glu-66 mutants from first principles using the Kirkwood-Fröhlich equation and discovered that staphylococcal nuclease has a naturally high dielectric constant ranging from 20 to 30. This high dielectric constant does not change significantly with the mutation of residue 66 or with the ionization of the mutated residues. Calculation of the spatial dependence of the dielectric constant for the microenvironment of residue-66 produces a value of about 10, which matches well with the apparent dielectric constant needed to reproduce pK(a) values from continuum electrostatic calculations. Our results suggest an alternative explanation that the high dielectric constant of staphylococcal nuclease is a property resulting from the intrinsic backbone fluctuations originating from its structural architecture.

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ˜2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins.

The Effect of Non-Coordinated Water in the Heme Pocket on the Ligand Binding Dynamics of Heme Proteins

Water molecules in internal protein cavities play fundamental roles in satisfying the H-bonding potentials of main chain atoms in turns, coils, and loops, determining the stability and rigidity of proteins, shifting the pKa values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers. Detecting these internal water molecules is sometimes obscured in x-ray crystallography due to positional disorder. We have developed a spectrokinetic assay that accurately detects the presence of a non-coordinated water molecule in the distal heme pocket of myoglobin and in a series of distal pocket mutants, including many where this water molecule is positionally disordered. We also have shown that this water plays a major role in determining the observed bimolecular recombination rate constant. We show that 1) this water molecule modulates the ligand binding dynamics of a series of H64, L29 and V68 mutants; 2) it plays the major role in the observed pH dependence of the CO recombination kinetics between pH 4 and 7 with the protonation of the distal histidine acting as a switch to change water occupancy; and 3) it may also modulate ligand binding dynamics in isolated hemoglobin chains, with the occupancy being larger in the alpha chains. Accurately measuring water occupancy in heme proteins answers crucial questions about water in apolar or slightly polar protein cavities and clarifies the role internal water molecules play in modulating protein function.

Vi -Value Analysis: A Pressure-Based Method for Mapping the Folding Transition State Ensemble of Proteins

Here we introduce an approach to mapping the folding transition state ensemble of proteins based on the pressure dependence of protein stability. Previously, we have shown that the activation volume for folding of wild type (WT) SNase is large and positive, and hence that the rate-limiting step in folding involves significant dehydration. In contrast, variants bearing buried ionizable residues at position 66 were shown recently to fold through a highly hydrated transition state ensemble (TSE). We present the effects on the pressure-jump folding kinetics of Lys substitutions in different internal positions throughout the structure. We calculate the Vi value of the variants as the activation volume for folding relative to that of the wild type. We find that the structure of the SNase WT includes part of the β-barrel and part of the first α-helix. The unique advantage of Vi-value analysis is that it conveys direct information about the state of hydration of the TSE, which has been recognized as a key factor in the protein folding transition.

Experimental measurement of the effective dielectric in the hydrophobic core of a protein

The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed p K a shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The p K a values of buried lysines were measured by difference potentiometry. The extent of coupling between the p K a and the global stability of the protein was evaluated by measuring p K a values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol −1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published p K a values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.