Browsing by Author "MacKenzie, Kevin R."
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Item Characterization of transmembrane domain interactions by the syndecan family of integral membrane proteins(2008) Dews, Ian Charles; MacKenzie, Kevin R.Protein-protein interactions between the transmembrane domains (TMDs) of integral membrane proteins have been increasingly implicated in contributing to biological function. In this thesis, I explore the strength, specificity and sequence dependence of interactions made by the TMDs of the syndecans, a family of four human cell adhesion molecules. Primary sequence alignment of all known syndecan TMDs reveals a completely conserved GxxxG dimerization motif. This motif has been shown to drive dimerization of many biological TMDs, and its strong conservation within the syndecan family would seem to suggest that all syndecans will display a common self-association phenotype. In contrast to this expectation, I show that the syndecan TMDs display a hierarchy of association phenotypes, with the syndecan-1 TMD showing very weak dimerization, the syndecan-3 and -4 TMDs showing strong dimerization, and the syndecan-2 TMD showing very strong dimerization. I further show that oligomerization of the syndecan TMDs depends upon the sequence element GxxxGxxxA, which is also conserved across all known syndecans. Using single and double point mutations, show that residue identities at two positions flanking the GxxxGxxxA motif combine to produce much of the difference in self-association strengths across syndecan paralogs. Residue identities at these intervening positions are different between paralogs but strongly conserved across orthologs, indicating an evolutionary pressure to maintain the hierarchy of association phenotypes. I further show that each syndecan TMD is capable of forming heteromeric complexes with at least two other paralogs and that these interactions are also supported by the GxxxG motif. The strength and stoichiometry of the heteromeric interactions are also paralog specific, meaning that residues in addition to the GxxxG motif are responsible for directing these interactions. These findings show that, although all syndecans possess a GxxxGxxxA sequence element that supports oligomerization, additional residues modulate the strength and stoichiometry of both homo- and heterotypic interactions. The strong conservation of residues that give rise to paralog specific homo- and heterotypic interactions suggests that the complexity of these interactions may play a role in mediating syndecan functions.Item Specialization of a calmodulin-like protein: Androcam adopts a single conformation over the entire physiological range of calcium(2010) Joshi, Mehul Kirit; MacKenzie, Kevin R.The ubiquitous and highly conserved calcium binding protein calmodulin exhibits structural plasticity, broad target binding specificity and the ability to tune its affinity for Ca2+ ion. Collectively, these properties enable calmodulin to transduce biological calcium signals to hundreds of downstream targets. Despite the versatility of calmodulin, metazoans express many essential calmodulin-like proteins that perform tissue specific functions. In this thesis, I have studied androcam, an essential protein in D.melanogaster that is 67% identical to calmodulin, to determine how its structure, Ca+2 binding and target recognition properties differ from those of calmodulin and contribute to its unique function. My NMR structures solved at high and low calcium show that unlike calmodulin, which switches its conformation in response to changes in [Ca+2], each lobe of androcam is locked in a single fold over the entire physiological range of [Ca+2]. The androcam C-lobe has two EF hands which each ligand a Ca+2 ion, and is structurally similar to calmodulin. However, it binds Ca+2 much tighter than calmodulin and is therefore constitutively present in the Ca+2 bound "open" conformation that potentiates interactions with hydrophobic targets. The N lobe of androcam does not bind Ca+2 at physiological concentrations but is well structured and adopts a "closed" conformation similar to Ca+2-free calmodulin that is not expected to bind a hydrophobic anchor. Consistent with these structural observations, chemical shift perturbation experiments show that androcam interacts with the unique 'Insert2' peptide of the biological target Myosin VI with its C lobe only, whereas calmodulin binds this target using both lobes. Our results indicate that the androcam sequence has been optimized by evolution starting from the highly versatile calmodulin sequence to adopt only one of the many conformations that calmodulin can sample. We propose that many other calmodulin-like proteins might have also evolved to be specialists for a unique functional state out of the plethora of conformations that calmodulin has been shown to populate.Item Specificity of membrane helix-helix interactions by mutagenesis and structural analysis(2008) Sulistijo, Endah Susilowati; MacKenzie, Kevin R.The activity of apoptosis protein BNIP3 has been associated with its ability to form homodimeric and heteromeric associations through its carboxy-terminal transmembrane domain (TMD), but little is known about the chemical or physical basis of these interactions. In this thesis, I describe two approaches to examine the sequence requirements for BNIP3 TMD dimerization and the properties that drive and stabilize this association. The first approach employs saturation mutagenesis to generate a library of single mutants in the context of a fusion protein construct and SDS-PAGE combined with Western blotting to characterize the mutant dimerization phenotypes. The mutagenesis data maps the BNIP3 TMD dimerization region and identifies five interacting residues for BNIP3 TMD dimerization: Ala176, Gly180, and Gly184 form tandem GxxxG motifs that allow close approach of the helix backbones, and His173 and Ser172 form inter-monomer hydrogen bonds. The mutagenesis data also show that the sequence context in which these five critical residues are embedded affects the strength of TMD helix-helix interactions because several mutations at or near the dimer interface that leave the small residues and the inter-monomer hydrogen bonding intact can abolish or profoundly lower dimerization. The second approach to the study of BNIP3 TMD dimerization involves determining the structure of the TMD dimer using solution NMR spectroscopy. Reconstitution of the BNIP3 TMD peptide in the non-ionic detergent dodecylphosphocholine (DPC) and addition of dipalmitoylphosphatidylcholine (DPPC) yields peptide spectra with excellent peak dispersion and resolution. This quality enables collection of chemical shift, J coupling, and NOE distance restraints, thus allowing determination of the BNIP3 TMD dimer structure. The NMR structure of the BNIP3 TMD dimer reveals the details of how the elements of the BNIP3 TMD sequence cooperate to support dimerization and provides a context to interpret the effects of the saturation mutagenesis results. Results from the saturation mutagenesis and structural analyses establish an understanding of the BNIP3 TMD dimerization and provide a framework for further studies of BNIP3, which include but are not limited to thermodynamic studies, functional analyses, and molecular dynamics modeling of the BNIP3 TMD associations.Item Stability and specificity of transmembrane domain self-association by mutagenesis and protein design(2009) Jaszewski, Todd Michael; MacKenzie, Kevin R.Stability and specificity of transmembrane domain self-association by In this thesis, I investigate the sequence dependence of homodimerization of the transmembrane domain of the pro-apoptotic C. elegans protein BNIP3. Using site directed mutagenesis and two assays for dimerization, I show that the tight association of the CeBNIP3 transmembrane domain relies on overlapping but distinct sets of residues depending on the assay: in membranes, the critical residues are N183xxSFxxxGxxxG 194, whereas in detergents, the key residues are S186FxxGxxxGxxxS 198. The small residue Ser 186, the bulky residue Phe 187, and small residues Gly 190 and Gly 194 play key roles in CeBNIP3 dimerization in both assays. However, CeBNIP3 TMD self-association in detergents, but not membranes, depends critically on Ser 198; self-association in lipid bilayers, but not detergents, depends on Asn 183. Comparison with the previously identified dimerization motif for the human BNIP3 ortholog (SHxxAlxxGlxxG) shows that the residues that drive CeBNIP3 dimerization in membranes are chemically similar to, but distinct from, those that drive HsBNIP3 association. To explore how interfacial BNIP3 residues determine dimer stability and specificity, I generated a combinatorial library from the CeBNIP3 and HsBNIP3 motifs, (STT)(H/N)xx(A/S)(I/F)xxG(I/A)xxG, and tested the hybrid sequences for dimerization. All combinations of interfacial residues support strong to extremely strong dimerization in membranes, suggesting that the two parental sequences adopt similar structures. Not all sequences form dimers in detergents, and dimerization propensity correlates weakly with sequence hydrophobicity. Manipulating the solvent conditions to enhance the hydrophobic effect increases dimerization of some sequences but not others. The CeBNIP3 and HsBNIP3 transmembrane domains form homodimers but not heterodimers in detergents. Hybrid motif sequences show differing propensities to form heterodimers with wildtype CeBNIP3 TMD and HsBNIP3 TMD: some hybrids discriminate, binding only one wildtype sequence, and some interact with both. My findings identify the sequence elements responsible for stability and specificity of BNIP3-type transmembrane domain dimerization. My results also show that the hydrophobicity of membrane spans strongly influences their behavior in detergent assays of protein-protein interactions. The demonstration that altering the aqueous solvent conditions can improve the stability of integral membrane proteins in detergents may be of general importance in membrane protein biochemistry.Item Structural basis for calmodulin-mediated regulation of the ryanodine receptor(2008) Kilpatrick, Adina Maximciuc; MacKenzie, Kevin R.We investigated the structural basis of calmodulin (CaM)-mediated regulation of the skeletal muscle ryanodine receptor (RyR1), a calcium channel that plays a key role in excitation-contraction coupling in muscle cells. In order to understand the complex interaction of CaM with this receptor, we pursued NMR and X-ray crystallographic studies of CaM/RyR1 peptide complexes both in the presence and in the absence of calcium. We have determined the 2.0 A crystal structure of Ca2+CaM in complex with a 30-residue peptide corresponding to the binding region for CaM on RyR1 (residues 3614 to 3643). The structure reveals that hydrophobic anchor residues in the target arranged in a novel '1-17' spacing allow each calmodulin lobe to interact with the peptide independently. Solution NMR 15N relaxation measurements and residual dipolar couplings confirm the structure of each calmodulin lobe and show that the complex undergoes segmental domain motion. Fluorescence measurements indicate that CaM binds with both domains to the 3614-3643 peptide, whereas if the second anchor is unavailable, CaM can bind without collapsing on the target. The independence of the two lobes of calmodulin offers a structural explanation for how other domains may compete for binding to this region to regulate the channel. NMR studies of these interaction partners indicate that the RyR1 target binds to only the C-lobe of CaM at low calcium concentrations, similar to other ion channels whose activity is modulated by CaM. The conformation of the C-lobe in the calcium-free complex closely resembles the one seen in the crystal structure of the calcium-loaded complex, suggesting that binding of the RyR1 peptide locks the C-domain of CaM in a conformation similar to that of the calcium-loaded protein. Comparison of the CaM/peptide complexes at low and high calcium concentrations provides a model for how CaM interacts with this region of RyR1: the C-lobe is constitutively tethered to the 3614-3643 target and is calcium-loaded even at low, resting calcium levels, whereas high calcium induces the N-lobe to bind to this region. In this way, the N-lobe of CaM acts as a Ca2+ sensor for RyR1 by switching between different binding sites on the receptor.Item Structure of androcam supports specialized interactions with myosin VI(National Academy of Sciences, 2012) Joshi, Mehul K.; Moran, Sean; Beckingham, Kathleen M.; MacKenzie, Kevin R.Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca2þ and is locked in a “closed” conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca2þ-regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin- mediated Ca2þ signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins.