Elena Atanasova

Calmodulin Wraps around Its Binding Domain in the Plasma Membrane Ca2+ Pump Anchored by a Novel 18-1 Motif

Using solution NMR spectroscopy, we obtained the structure of Ca2+-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca2+-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM·C20 complex with this holoCaM·C28 complex supports the idea that the initial binding step is represented by (holoCaM·C20) and the final bound complex by (holoCaM·C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an αD-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

Novel Messenger RNA and Alternative Promoter for Murine Acetylcholinesterase

A portion of the 5′-flanking region of murine acetylcholinesterase was cloned from genomic DNA by 5′-rapid amplification of genomic ends, identified in a mouse genomic library, and sequenced. Multiple potential binding sites for universal and tissue-specific transcription factors were suggestive of a promoter region within this DNA sequence. Potential promoter activity was confirmed by coupling the new sequence to the open reading frame of a luciferase reporter gene in transient expression experiments with nerve and muscle cells. 5′-Rapid amplification of cDNA ends with templates from multiple sources revealed a novel transcription start site (at position −626, relative to translation start), located 32 bases downstream from a TATAA sequence. This start site appeared to mark a novel exon (1a) comprising 291 base pairs between positions −335 and −626, relative to the translation start. Supporting this conclusion, polymerase chain reactions with cDNA from mouse brain, heart, and other tissues, consistently amplified a transcript containing the exon 1a sequence fused to the invariant sequence beginning at position −22 in exon 2, but lacking exon 1. Northern blot analyses confirmed the in vivo expression of exon 1a-containing transcripts, especially in heart, brain, liver, and kidney. These results indicate that the murine acetylcholinesterase gene has a functioning alternative promoter that may influence expression of acetylcholinesterase in certain tissues.

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Background: The calcium-binding protein DREAM/KChIP3 binds DNA and other proteins to regulate neuronal function. Results: DREAM/KChIP3 binds the EF-hand protein, calmodulin, in the presence but not in the absence of calcium. Calcium-bound DREAM/KChIP3 enhances calmodulin-dependent calcineurin activity. Conclusion: DREAM/KChIP3 binds and regulates calmodulin activity. Significance: DREAM/KChIP3 heterodimerizes with the EF-hand protein, calmodulin, and regulates calmodulin activation of calcineurin. Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca2+) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca2+, DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca2+. In the absence of Ca2+, DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca2+-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μm as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional 1H,15N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to 15N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca2+-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca2+ also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH2-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

Structural dependencies of protein backbone 2JNC′ couplings

Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone 2JNC′ couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental 2JNC′ couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated 2JNC′ couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H‐bond strengths derived from h3JNC′ couplings, provides useful insight into the overall energy profile of the protein backbone in solution.

Solvent‐induced differentiation of protein backbone hydrogen bonds in calmodulin

In apo and holoCaM, almost half of the hydrogen bonds (H‐bonds) at the protein backbone expected from the corresponding NMR or X‐ray structures were not detected by h3 JNC′ couplings. The paucity of the h3 JNC′ couplings was considered in terms of dynamic features of these structures. We examined a set of seven proteins and found that protein‐backbone H‐bonds form two groups according to the h3 JNC′ couplings measured in solution. H‐bonds that have h3 JNC′ couplings above the threshold of 0.2 Hz show distance/angle correlation among the H‐bond geometrical parameters, and appear to be supported by the backbone dynamics in solution. The other H‐bonds have no such correlation; they populate the water‐exposed and flexible regions of proteins, including many of the CaM helices. The observed differentiation in a dynamical behavior of backbone H‐bonds in apo and holoCaM appears to be related to protein functions.

Antibody-Targeted Chemotherapy for the Treatment of Melanoma.

Antibody-directed chemotherapy (ADC) offers an advantage over conventional chemotherapy because it provides antibody-directed targeting, with resultant improvement in therapeutic efficacy and reduced toxicity. Despite extensive research, with notable exceptions, broad clinical application of ADC remains elusive; major hurdles include the instability of antibody-chemotherapy linkers and reduced tumor toxicity of the chemotherapy when bound to the antibody. To address these challenges, we have developed a platform technology that utilizes the nab-paclitaxel formulation of paclitaxel, Abraxane, in which hydrophobic paclitaxel is suspended in 130-nm albumin nanoparticles and thus made water-soluble. We have developed a method to noncovalently coat the Abraxane nanoparticle with recombinant mAbs (anti-VEGF, bevacizumab) and guide Abraxane delivery into tumors in a preclinical model of human A375 melanoma. Here, we define the binding characteristics of bevacizumab and Abraxane, demonstrate that the chemotherapy agent retains its cytotoxic effect, while the antibody maintains the ability to bind its ligand when the two are present in a single nanoparticle (AB160), and show that the nanoparticle yields improved antitumor efficacy in a preclinical human melanoma xenograft model. Further data suggest that numerous therapeutic monoclonal IgG1 antibodies may be utilized in this platform, which has implications for many solid and hematologic malignancies. Cancer Res; 76(13); 3954-64. ©2016 AACR.

Photophysics of EGFP (E222H) Mutant, with Comparisons to Model Chromophores: Excited State pK’s, Progressions, Quenching and Exciton Interaction

AbstractA novel version of the well-known and commercially successful Green Fluorescent Protein (GFP) variant known as EGFP, with an introduced E222H mutation, was produced in this laboratory. Given the current state of hypotheses about the role of glutamate 222, and the observed dominance of the phenolate absorption with an E222H variant observed from earlier study, the new mutant was considered a natural choice to investigate more fully the acid-base behavior of the chromophore in absorption and fluorescence. The bulk of this investigation concerns fitting the excitation, emission and absorption spectra to vibrational progressions of a novel ‘q-deformed’ type at various values of pH, and protein concentration. From these data, and from temperature-dependent fluorescence lifetime data and other experiments (with lanthanide doped gels into which H/EGFP is embedded), we construct a picture of excited inter- state conversion mechanisms, and quenching mechanisms, that attempts to explain many features of the GFP system. Graphical AbstractHypothetical proton current loop (orange) upon excitation; electron motion in purple H/EGFP. Solid boxes about waters project toward viewer, dashed boxes project away