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Structure and Dynamics Study of Cu Transporting Atpase by NMR

Title: Structure and Dynamics Study of Cu Transporting Atpase by NMR.
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Name(s): Meng, Dan, Ph. D., author
Brüschweiler, Rafael P., professor co-directing dissertation
Logan, Timothy M., 1961-, professor co-directing dissertation
Taylor, Kenneth A., university representative
Taylor, Kenneth A., university representative
Li, Hong, committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Department of Chemistry and Biochemistry, degree granting department
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2014
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (107 pages)
Language(s): English
Abstract/Description: Copper transporting ATPase (Cu-ATPase) is a member of the P1B-subfamily of P-type ATPases, which catalyzes ATP-dependent copper delivery across cellular membranes. The energy derived from ATP is used in the active transport of Cu+ against a concentration gradient across the membrane. During this process, an invariant aspartate residue is auto-phosphorylated and dephosphorylated. Cu-ATPase shares the same type of basic architecture with other P-type ATPases, such as Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SERCA). This family of ATPases all possesses three cytoplasmic domains that are the nucleotide-binding (N) domain, the phosphorylation (P) domain, and the actuator (A) domain, as well as the transmembrane domain. Cu-ATPase differs from SERCA by having one or several N-terminal metal-binding domains. Here, NMR is used to characterize the in solution structure and dynamics of the N and P domains (A387-N675, 31 kDa) of Archaeoglobus fulgidus CopA (CopA_NPwt). Our NMR results suggest that the P domain of CopA_NPwt is undergoing conformational exchange in both the apo and the AMPPCP bound state. Disulfide linking of the N and C terminal helices of CopA_NPwt helps stabilize the P domain resulting in a more organized state (CopA_NPss). CopA_NPss is generally very rigid in both N and P domains on the fast timescale, with the exception of a few flexible loops. The inter-domain orientation of CopA_NPss was determined by using residual dipolar couplings, which report the average population in solution. The N and P domains close up upon AMPPCP binding, which results in a highly compact conformation in the transition state, until phosphorylation occurs that opens up the N and P domains. Moreover, the role of the conserved 548DXXK motif in the hinge of CopA_NPss was explored by studying the structural and dynamics effect of the D548N mutation. This mutation does not affect the inter-domain orientation or the inter-domain flexibility of CopA_NPss, but the D548N mutation resulted in a 5-fold increase of the binding affinity for AMPPCP. The communication mechanism between the phosphorylation site and the cation binding site of the transmembrane helices was explored. Cation binding to the transmembrane helices drives the stabilization of the P domain, resulting in a functionally efficient phosphorylation pocket wedged between the N and P domains. Phosphorylation of the invariant Asp residue stabilizes a more open inter-domain orientation between the N and P domains, which gives access to the A domain and its dephosphorylation peptide region to undo phosphorylation while opening the transmembrane region enabling ion transport across the membrane. This model suggests how the allosteric communication could be achieved between the phosphorylation site and the ligand-binding site of the transmembrane domain, which can be tested and refined through future experiments both in solution and in crystals.
Identifier: FSU_migr_etd-9217 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Fall Semester, 2014.
Date of Defense: September 8, 2014.
Bibliography Note: Includes bibliographical references.
Advisory Committee: Rafael P. Brüschweiler, Professor Co-Directing Dissertation; Timothy M. Logan, Professor Co-Directing Dissertation; Kenneth A. Taylor, University Representative; Hong Li, Committee Member.
Subject(s): Biochemistry
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_migr_etd-9217
Owner Institution: FSU

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Meng, D. (2014). Structure and Dynamics Study of Cu Transporting Atpase by NMR. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-9217