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Macromolecular Structure: from peptides to polyvalent proteins

Stachowski, Kye
http://rave.ohiolink.edu/etdc/view?acc_num=osu1626691913293814

Abstract Details

2021, Doctor of Philosophy, Ohio State University, Biochemistry.
Understanding the structure and function of macromolecules and their performative complexes is indispensable when developing novel treatments for disease. Nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) provide powerful insights into macromolecular structure and dynamics and thereby harbor the potential for aiding the development of much needed therapeutics. The studies presented here describe the application of NMR spectroscopy and cryo-EM to characterize the structure and/or dynamics of three distinct biochemical entities with fundamental biological implications. These targets include cyclic peptides being developed as cancer therapies, a homo-oligomeric ring protein that serves as a model system for understanding allosteric regulation, and a recombinase that selectively binds and becomes activated to site-specifically cleave DNA. Cyclic peptides are capable of binding to challenging molecular targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to the intracellular environment because of poor membrane permeability. In chapter 2, I describe my work to characterize a pair of conformationally constrained cyclic cell-penetrating peptides (CPP) containing a D-Pro-L-Pro motif, beginning with cyclo(AFΦrpPRRFQ) (where Φ is L-naphthylalanine, r is D-arginine, and p is D-proline). The structural constraints provided by cyclization and the D-Pro-L-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied by my collaborators to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(YpVNFΦrpPRR) (where Yp is L-phosphotyrosine), against the Grb2 SH2 domain, a key mediator in Ras activation. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the initial cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant β-hairpin character surrounding the D-Pro-L-Pro motif. These results demonstrate that cyclo(AFΦrpPRRFQ) is an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain). In nature, ring shaped holo-oligomeric proteins allosterically control their function to perform many necessary life functions, such as helicases translocating along strands of nucleic acids by allosterically binding and hydrolysis of ATP. In Chapter 3, I describe the use of Cryo-EM to study the structural effects of tryptophan binding to the ring protein TRAP, trp RNA-binding attenuation protein. TRAP, isolated from Bacillus halodurans, contains 12 binding sites for tryptophan and a specific RNA motif. When the TRAP ring reaches a threshold of bound tryptophan (Trp) molecules, the ring becomes activated to bind mRNA sequences in the 5’ untranslated region of the trp operon and thereby inhibit translation of enzymes responsible for tryptophan biosynthesis. The Trp binding sites are located at the interface of two adjacent protomers, and previous work on Bacillus stearothermophilus TRAP has shown that the affinity of Trp for a site is influenced by whether the neighboring sites are empty or bound to Trp. This nearest neighbor model was shown to explain variable temperature binding data measured by ITC and mass spectrometry. We have applied Cryo-EM to understand how allosteric communication between two sites in TRAP is realized through organizational behavior at unoccupied binding sites. My collaborators developed a linked dimer TRAP system (dTRAP) that allows for study of an every-other-site occupied configuration of TRAP. Negative stain EM data provided models indicating that the dTRAP system still forms rings, and that the apo form of dTRAP is more disordered than the holo form (similar to the non-linked version). Cryo-EM structures of the apo and holo dTRAP have been solved, and I describe work towards structure determination of the half-loaded dTRAP. Mechanistic understanding of the structural basis for DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes (intasomes). Those studies have suggested that conformational changes and DNA bending in presynaptic complexes underlie the assembly and activation mechanism of Cre recombinase. Solution nuclear magnetic resonance (NMR) spectroscopy revealed how the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein–protein interactions, adopts an alternative autoinhibitory conformation, wherein it packs in cis over the protein DNA binding surface and active site in the absence of DNA. Binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that enables protein–protein interactions required for assembly of recombinogenic Cre intasomes. Furthermore, we used protein engineering to isolate Cre-loxP and Cre2-loxP complexes, and determined the structures of assembly intermediates (monomer, dimer, tetramer) using cryo-EM to resolutions of 3.9 Å, 4.5 Å and 3.2 Å, respectively. We found that as Cre assembles into an activated complex, the bend of the loxP DNA site becomes more pronounced as each intermediate is reached, and deformations of the loxP DNA are key selectivity determinants of Cre. The progressive DNA bending is accompanied by increased protein-protein interactions. Our work shows how tetramerization is required for Cre to become activated to recombine DNA. 3D variability analysis uncovered conformational sampling within the Cre tetrameric complex that reveals how the protein-protein interfaces of the tetramer are important for activation of Cre and selectivity for loxP DNA. These new insights could prove useful in design of new Cre variants with engineered site-specificity and improved recombination efficiency. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.
Mark Foster (Advisor)
Thomas Magliery (Committee Member)
Charles Bell (Committee Member)
206 p.
Biochemistry
Cre recombinase, Cryo-EM, cryo electron microscopy, TRAP, trp, trp RNA-binding attenuation protein, cyclic peptide, cell-penetrating peptide, CPP, macrocycle, DNA binding, structural biology, NMR, PRE

Recommended Citations

Citations

  • Stachowski, K. (2021). Macromolecular Structure: from peptides to polyvalent proteins [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1626691913293814

    APA Style (7th edition)

  • Stachowski, Kye. Macromolecular Structure: from peptides to polyvalent proteins. 2021. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1626691913293814.

    MLA Style (8th edition)

  • Stachowski, Kye. "Macromolecular Structure: from peptides to polyvalent proteins." Doctoral dissertation, Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu1626691913293814

    Chicago Manual of Style (17th edition)

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