Tatyana I. Igumenova

Dynamics and Membrane Interactions of Protein Kinase C

Protein kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. Significant research efforts have been directed at understanding the structure, function, and regulatory modes of the enzyme since its discovery and identification as the first receptor for tumor-promoting phorbol esters. The activation of PKC involves a transition from the cytosolic autoinhibited latent form to the membrane-associated active form. The membrane recruitment step is accompanied by the conformational rearrangement of the enzyme, which relieves autoinhibitory interactions and thereby allows PKC to phosphorylate its targets. The multidomain structure and intrinsic flexibility of PKC present remarkable challenges and opportunities for the biophysical and structural biology studies of this class of enzymes and their interactions with membranes, the major focus of this Current Topic. I will highlight the recent advances in the field, outline the current challenges, and identify areas where biophysics and structural biology approaches can provide insight into the isoenzyme-specific regulation of PKC activity.

Pb2+ as modulator of protein-membrane interactions

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.

Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

A Sec14-nodulin protein model is used to identify the nodulin domain as a novel phosphoinositide effector module with a role in controlling lateral organization of phosphoinositide. The domain organization of Sec14-nodulin proteins suggests a versatile principle for the bit mapping of membrane surfaces into high-definition lipid-signaling screens.

Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα.

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a >100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.

The Structural Basis of Iron Sensing by the Human F-box Protein FBXL5

Iron is an essential chemical element for all forms of life. It serves as a cofactor for many proteins and enzymes involved in oxygen transport, energy metabolism and DNA synthesis[1, 2]. Mammalian cells need to maintain a sufficient amount of iron to support the synthesis of proteins that require iron as a co-factor. Iron is imported into the cells through the circulating iron transporter transferrin[3, 4]. Iron-loaded transferrin binds to cell surface transferrin receptor, resulting in the endocytosis of transferrin and delivery of the iron cargo into the cells[4]. Excess iron in the cells is stored in the cytosolic protein ferritin[4]. Iron regulatory proteins IRP1 and IRP2 maintain the homeostasis of iron through post-translational regulation of the expression of transferrin receptor and ferritin[2, 5]. In iron-replete cells IRP2 is ubiquitinated and degraded by the proteasome. The F-box and leucine-rich repeat containing protein FBXL5 serves as a cytosolic iron sensor that regulates the ubiquitination of IRP2 by the SKP1-CUL1-FBXL5 (SCF) E3 ubiquitin ligase complex[6, 7]. FBXL5 also plays critical roles in sensing oxygen in the cytosol[6, 7]. Knock out of FBXL5 in mice result in embryonic mortality due to excess accumulation of iron[8].

Reactive Cysteine in the Structural Zn2+ Site of the C1B Domain from PKCα

Structural cysteine-rich Zn(2+) sites that stabilize protein folds are considered to be unreactive. In this article, we identified a reactive cysteine residue, Cys151, in a treble-clef zinc finger with a Cys(3)His coordination sphere. The protein in question is the C1B domain of Protein Kinase Cα (PKCα). Mass-tagging cysteine assays of several C1B variants were employed to ascertain the site specificity of the covalent modification. The reactivity of Cys151 in C1B also manifests itself in the structural dynamics of the Zn(2+) coordination sphere where the Sγ of Cys151 alternates between the Zn(2+)-bound thiolate and free thiol states. We used NMR-detected pH titrations, ZZ-exchange spectroscopy, and residual dipolar coupling (RDC)-driven structure refinement to characterize the two exchanging conformations of C1B that differ in zinc coordination. Our data suggest that Cys151 serves as an entry point for the reactive oxygen species that activate PKCα in a process involving Zn(2+) release.

The C-Terminal V5 Domain of Protein Kinase Cα Is Intrinsically Disordered, with Propensity to Associate with a Membrane Mimetic

The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the “cis-trans” conformer of the Thr638-Pro639-Pro640 turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved “NFD” motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.

Interfacial partitioning of a loop hinge residue contributes to diacylglycerol affinity of conserved region 1 domains.

Background: The activity of protein kinase C isoenzymes (PKCs) is regulated by diacylglycerol (DAG). Results: Aromatic residue that tunes DAG affinity influences the recruitment of the conserved region 1 (C1) regulatory domain to a membrane mimic. Conclusion: Membrane pre-association step contributes to the affinity of C1 to diacylglycerol. Significance: This work offers insight into the origin of differential DAG affinities in PKCs. Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826–830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities.

Synergistic effect of Pb(2+) and phosphatidylinositol 4,5-bisphosphate on C2 domain-membrane interactions.

Ca(2+)-responsive C2 domains are peripheral membrane modules that target their host proteins to anionic membranes upon binding Ca(2+) ions. Several C2 domain-containing proteins, such as protein kinase C isoenzymes (PKCs), have been identified as molecular targets of Pb(2+), a known environmental toxin. We demonstrated previously that the C2 domain from PKCα (C2α) binds Pb(2+) with high affinity and undergoes membrane insertion in the Pb(2+)-complexed form. The objective of this work was to determine the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) on the C2α-Pb(2+) interactions. Using nuclear magnetic resonance (NMR) experiments, we show that Pb(2+) and PIP(2) synergistically enhance each others affinity for C2α. Moreover, the affinity of C2α for PIP(2) increases upon progressive saturation of the metal-binding sites. Combining the NMR data with the results of protein-to-membrane Förster resonance energy transfer and vesicle sedimentation experiments, we demonstrate that PIP(2) can influence two aspects of C2α-Pb(2+)-membrane interactions: the affinity of C2α for Pb(2+) and the association of Pb(2+) with the anionic sites on the membrane. Both factors may contribute to the toxic effect of Pb(2+) resulting from the aberrant modulation of PKCα activity. Finally, we propose a mechanism for Pb(2+) outcompeting Ca(2+) from membrane-bound C2α.

Cd2+ as a Ca2+ surrogate in protein-membrane interactions: isostructural but not isofunctional.

Due to its favorable spectroscopic properties, Cd(2+) is frequently used as a probe of Ca(2+) sites in proteins. We investigate the ability of Cd(2+) to act as a structural and functional surrogate of Ca(2+) in protein-membrane interactions. C2 domain from protein kinase Cα (C2α) was chosen as a paradigm for the Ca(2+)-dependent phosphatidylserine-binding peripheral membrane domains. We identified the Cd(2+)-binding sites of C2α using NMR spectroscopy, determined the 1.6 Å crystal structure of Cd(2+)-bound C2α, and characterized metal-ion-dependent interactions between C2α and phospholipid membranes using fluorescence spectroscopy and ultracentrifugation experiments. We show that Cd(2+) forms a tight complex with the membrane-binding loops of C2α but is unable to support its membrane-binding function. This is in sharp contrast with Pb(2+), which is almost as effective as Ca(2+) in driving the C2α-membrane association process. Our results provide the first direct evidence for the specific role of divalent metal ions in mediating protein-membrane interactions, have important implications for metal substitution studies in proteins, and illustrate the potential diversity of functional responses caused by toxic metal ions.