Proline substitution of dimer interface ß-strand residues as a strategy for the design of functional monomeric proteins.

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface ß-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface ß-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface ß-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface ß-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.

Identification of functionally important residues/domains in membrane proteins using an evolutionary approach coupled with systematic mutational analysis.

Structure-function studies of membrane proteins present a unique challenge to researchers due to the numerous technical difficulties associated with their expression, purification and structural characterization. In the absence of structural information, rational identification of putative functionally important residues/regions is difficult. Phylogenetic relationships could provide valuable information about the functional significance of a particular residue or region of a membrane protein. Evolutionary Trace (ET) analysis is a method developed to utilize this phylogenetic information to predict functional sites in proteins. In this method, residues are ranked according to conservation or divergence through evolution, based on the hypothesis that mutations at key positions should coincide with functional evolutionary divergences. This information can be used as the basis for a systematic mutational analysis of identified residues, leading to the identification of functionally important residues and/or domains in membrane proteins, in the absence of structural information apart from the primary amino acid sequence. This approach is potentially useful in the context of the auditory system, as several key processes in audition involve the action of membrane proteins, many of which are novel and not well characterized structurally or functionally to date.

Essential helix interactions in the anion transporter domain of prestin revealed by evolutionary trace analysis.

Prestin, a member of the SLC26A family of anion transporters, is a polytopic membrane protein found in outer hair cells (OHCs) of the mammalian cochlea. Prestin is an essential component of the membrane-based motor that enhances electromotility of OHCs and contributes to frequency sensitivity and selectivity in mammalian hearing. Mammalian cells expressing prestin display a nonlinear capacitance (NLC), widely accepted as the electrical signature of electromotility. The associated charge movement requires intracellular anions reflecting the membership of prestin in the SLC26A family. We used the computational approach of evolutionary trace analysis to identify candidate functional (trace) residues in prestin for mutational studies. We created a panel of mutations at each trace residue and determined membrane expression and nonlinear capacitance associated with each mutant. We observe that several residue substitutions near the conserved sulfate transporter domain of prestin either greatly reduce or eliminate NLC, and the effect is dependent on the size of the substituted residue. These data suggest that packing of helices and interactions between residues surrounding the "sulfate transporter motif" is essential for normal prestin activity.

Functional expression and microdomain localization of prestin in cultured cells.

INTRODUCTION: Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. METHODS: We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. RESULTS & DISCUSSION: Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. CONCLUSION: Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.

Structural basis of chemokine receptor function--a model for binding affinity and ligand selectivity.

Chemokine receptors play fundamental roles in human physiology from embryogenesis to inflammatory response. The receptors belong to the G-protein coupled receptor class, and are activated by chemokine ligands with a range of specificities and affinities that result in a complicated network of interactions. The molecular basis for function is largely a black box, and can be directly attributed to the lack of structural information on the receptors. Studies to date indicate that function can be best described by a two-site model, that involves interactions between the receptor N-domain and ligand N-terminal loop residues (site-I), and between receptor extracellular loop and the ligand N-terminal residues (site-II). In this review, we describe how the two-site model could modulate binding affinity and ligand selectivity, and also highlight some of the unique chemokine receptor features, and their role in function.

Membrane prestin expression correlates with the magnitude of prestin-associated charge movement.

Full expression of electromotility, generation of non-linear capacitance (NLC), and high-acuity mammalian hearing require prestin function in the lateral wall of cochlear outer hair cells (OHCs). Estimates of the number of prestin molecules in the OHC membrane vary, and a consensus has not emerged about the correlation between prestin expression and prestin-associated charge movement in the OHC. Using an inducible prestin-expressing cell line, we demonstrate that the charge density, but not the voltage at peak capacitance, directly correlates with the amount of prestin in the plasma membrane. This correlation is evident in studies involving a controlled increase of prestin expression with time after induction and inducer dose-response. Conversely, membrane prestin levels and charge density gradually decline together following the reduction of prestin levels from a steady state by removal of the inducer. Thus, charge density directly correlates with the level of membrane prestin expression, whereas changing membrane levels of prestin have no effect on the voltage at peak capacitance in this inducible prestin-expressing cell line.

The monomer-dimer equilibrium and glycosaminoglycan interactions of chemokine CXCL8 regulate tissue-specific neutrophil recruitment.

Chemokines exert their function by binding the GPCR class of receptors on leukocytes and cell surface GAGs in target tissues. Most chemokines reversibly exist as monomers and dimers, but very little is known regarding the molecular mechanisms by which the monomer-dimer equilibrium modulates in vivo function. For the chemokine CXCL8, we recently showed in a mouse lung model that monomers and dimers are active and that the monomer-dimer equilibrium of the WT plays a crucial role in regulating neutrophil recruitment. In this study, we show that monomers and dimers are also active in the mouse peritoneum but that the role of monomer-dimer equilibrium is distinctly different between these tissues and that mutations in GAG-binding residues render CXCL8 less active in the peritoneum but more active in the lung. We propose that tissue-specific differences in chemokine gradient formation, resulting from tissue-specific differences in GAG interactions, are responsible for the observed differences in neutrophil recruitment. Our observation of differential roles played by the CXCL8 monomer-dimer equilibrium and GAG interactions in different tissues is novel and reveals an additional level of complexity of how chemokine dimerization regulates in vivo recruitment.

Monomeric and dimeric CXCL8 are both essential for in vivo neutrophil recruitment.

Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a 'trapped' non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.

Glycosylation regulates prestin cellular activity.

Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin- and caveolin-dependent mechanisms.

Membrane composition modulates prestin-associated charge movement.

The lateral membrane of the cochlear outer hair cell (OHC) is the site of a membrane-based motor that powers OHC electromotility, enabling amplification and fine-tuning of auditory signals. The OHC membrane protein prestin plays a central role in this process. We have previously shown that membrane cholesterol modulates the peak voltage of prestin-associated nonlinear capacitance in vivo and in vitro. The present study explores the effects of membrane cholesterol and docosahexaenoic acid content on the peak and magnitude of prestin-associated charge movement in a human embryonic kidney (HEK 293) cell model. Increasing membrane cholesterol results in a hyperpolarizing shift in the peak voltage of the nonlinear capacitance (Vpkc) and a decrease in the total charge movement. Both measures depend linearly on membrane cholesterol concentration. Incubation of cholesterol-loaded cells in cholesterol-free media partially restores the Vpkc toward normal values but does not have a compensatory effect on the total charge movement. Decreasing membrane cholesterol results in a depolarizing shift in Vpkc that is restored toward normal values upon incubation in cholesterol-free media. However, cholesterol depletion does not alter the magnitude of charge movement. In contrast, increasing membrane docosahexaenoic acid results in a hyperpolarizing shift in Vpkc that is accompanied by an increase in total charge movement. Our results quantify the relation between membrane cholesterol concentration and prestin-associated charge movement and enhance our understanding of how membrane composition modulates prestin function.

Role of intramolecular disulfides in stability and structure of a noncovalent homodimer.

The importance of intramolecular disulfides in a noncovalent dimeric protein interleukin-8 (IL-8) has been studied by replacing cysteines in each of the two disulfide pairs with alpha-aminobutyric acid (CH(2)-SH --> CH(2)-CH(3)). Both disulfide mutants are less stable and exist as molten globules in the monomeric state. Interestingly, both mutants dimerize, though with slightly lower affinities compared to the native protein. NMR studies suggest a molten globule-like structure also in the dimeric state. Structures, sequence analysis, and mutagenesis studies have shown that the conserved hydrophobic residues are packed against each other in the protein core and that H bonding and van der Waals interactions stabilize the dimer interface. Deleting either disulfide in IL-8 results in substantial loss in receptor activity, indicating that both disulfides are critical for function in the folded protein. These data together suggest that the packing interactions of the hydrophobic core determine IL-8 monomer fold, that disulfides play only a marginal role in dimer formation, and that the stability imparted by the disulfides is intimately coupled to fold and function.

Prestin modulates mechanics and electromechanical force of the plasma membrane.

The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.

Effects of prestin on membrane mechanics and electromechanics.

The electromotility of cochlear outer hair cells (OHCs) is a required process for normal hearing, and involves a membrane-based mechanism in which the transmembrane protein, prestin, plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanics of the cell membrane using membrane tethers formed from human embryonic kidney cells. Our results suggest that prestin appears to change membrane tension and amplify electrically-evoked force generation, while a single point mutation of alanine to tryptophan in prestin reduces electrically-evoked force generation without affecting the membrane tension. We propose that prestin and membrane work in synergy to produce the electrical and mechanical changes that are required during OHC electromotility.

Tuning of the outer hair cell motor by membrane cholesterol.

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Novel use of an osmolyte to dissect multiple thermodynamic linkages in a chemokine ligand-receptor system.

We have used trimethylamine N-oxide (TMAO), a protecting osmolyte, to dissect the complex thermodynamic linkages involved in the interaction between the chemokine interleukin-8 (IL-8) and the N-domain of its receptor CXCR1. Our results show that TMAO induces folding in the CXCR1 receptor N-domain and that the N-domain upon folding binds ligand with higher affinity. This represents, to our knowledge, the smallest domain that has been shown to be folded in osmolyte. Using the phase diagram method to analyze this thermodynamic relationship graphically, we also observe that TMAO favors ligand dimerization and that the dimeric ligand binds the receptor domain with lower affinity. We have thus been able to dissect coupling among three distinct processes, receptor domain folding, ligand dimerization, and ligand-receptor domain binding in this chemokine-receptor system. We also observe that the affinity of the related chemokine, melanoma growth stimulatory activity (MGSA), increases concurrent with N-domain folding similar to IL-8 but shows more profound differences on ligand dimerization. These studies establish a novel and innovative use of osmolytes to dissect linkages among different processes and exploit the phase diagram as a tool to graphically represent and dissect complex thermodynamic relationships in biological systems. On the basis of our observations and earlier work, we discuss the relevance of ligand dimerization in chemokine regulation.

Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain.

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (approximately 1 microM versus approximately 20 microM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (approximately 3 microM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.

Role of His-16 in turnover of T4 pyrimidine dimer glycosylase.

Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.

Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein.

The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.