Brett M. Kroncke

DEER EPR Measurements for Membrane Protein Structures via Bifunctional Spin Labels and Lipodisq Nanoparticles

Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to those of globular proteins. In this study, we employed a combination of three recently developed methodologies, (1) bifunctional spin labels (BSL), (2) SMA-Lipodisq nanoparticles, and (3) Q band pulsed EPR measurements, to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated an ∼2-fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to those of proteoliposomes and narrower distance distributions for the BSL when compared to those of the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.

MAPK Phosphorylation of Connexin 43 Promotes Binding of Cyclin E and Smooth Muscle Cell Proliferation

Rationale: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. Objective: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. Methods and Results: We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE−/−) mice and in C57Bl/6 mice treated with platelet-derived growth factor–BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43CT) and produced null phosphorylation Ser>Ala (Cx43MK4A/Cx43CTMK4A) and phospho-mimetic Ser>Asp (Cx43MK4D/Cx43CTMK4D) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43+/+) and Cx43 null (Cx43−/−) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury. Conclusions: We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.

Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin labels intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.

Reversible folding of human peripheral myelin protein 22, a tetraspan membrane protein.

Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22.

Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes

KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.

Structure of the neisserial outer membrane protein opa60: loop flexibility essential to receptor recognition and bacterial engulfment.

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.

Purification and structural study of the voltage-sensor domain of the human KCNQ1 potassium ion channel.

KCNQ1 (also known as KV7.1 or KVLQT1) is a voltage-gated potassium channel modulated by members of the KCNE protein family. Among multiple functions, KCNQ1 plays a critical role in the cardiac action potential. This channel is also subject to inherited mutations that cause certain cardiac arrhythmias and deafness. In this study, we report the overexpression, purification, and preliminary structural characterization of the voltage-sensor domain (VSD) of human KCNQ1 (Q1-VSD). Q1-VSD was expressed in Escherichia coli and purified into lyso-palmitoylphosphatidylglycerol micelles, conditions under which this tetraspan membrane protein yields excellent nuclear magnetic resonance (NMR) spectra. NMR studies reveal that Q1-VSD shares a common overall topology with other channel VSDs, with an S0 helix followed by transmembrane helices S1–S4. The exact sequential locations of the helical spans do, however, show significant variations from those of the homologous segments of previously characterized VSDs. The S4 segment of Q1-VSD was seen to be α-helical (with no 310 component) and underwent rapid backbone amide H–D exchange over most of its length. These results lay the foundation for more advanced structural studies and can be used to generate testable hypotheses for future structure–function experiments.

Personalized Biochemistry and Biophysics

Whole human genome sequencing of individuals is becoming rapid and inexpensive, enabling new strategies for using personal genome information to help diagnose, treat, and even prevent human disorders for which genetic variations are causative or are known to be risk factors. Many of the exploding number of newly discovered genetic variations alter the structure, function, dynamics, stability, and/or interactions of specific proteins and RNA molecules. Accordingly, there are a host of opportunities for biochemists and biophysicists to participate in (1) developing tools to allow accurate and sometimes medically actionable assessment of the potential pathogenicity of individual variations and (2) establishing the mechanistic linkage between pathogenic variations and their physiological consequences, providing a rational basis for treatment or preventive care. In this review, we provide an overview of these opportunities and their associated challenges in light of the current status of genomic science and personalized medicine, the latter often termed precision medicine.

Structural basis for KCNE3 modulation of potassium recycling in epithelia

KCNE3 modulates the KCNQ1 K+ channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state. The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”

Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability

There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < −0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.