Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies

Sharma, Alok; Sawhney, Paramvir; Memisoglu, Gonen; Rigby, Alan C.

doi:10.1016/j.pep.2011.09.013

Cited by 2 publications

(11 citation statements)

References 41 publications

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“…13 C/ 15 N-or 15 N-labeled CC MBS polypeptides of Ͼ96% apparent purity migrated as a single homodimeric peak on SEC (Fig. 1B), consistent with previous results (16). Fig.…”

Section: Protein Purification and Nmr Spectroscopysupporting

confidence: 91%

“…However, the absence of 3D structural information for CC MBS and lack of knowledge of the core residues involved in its binding to PKG-I␣ have limited our structural understanding of the CC MBS⅐LZ PKG-I␣ complex. We and others have demonstrated that CC MBS is a folded homodimer in solution (9,14,16) and exhibits a well-dispersed 2D 1 H- 15 N HSQC spectrum (16). We also reported 1 H, 13 C, and 15 N NMR assignments for CC MBS and identified critical amino acid residues involved in formation of the ␣-helical coiled-coil region of CC MBS (17).…”

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confidence: 52%

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NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function

Sharma

Birrane

Anklin

et al. 2017

Journal of Biological Chemistry

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Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-1α has remained limited. Here, we report the 3D NMR solution structure of homodimeric CC MBS in which amino acids 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at and heptad positions. Using NMR chemical-shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation enhancement- and CSP-NMR-guided HADDOCK modeling of the dimer-dimer interface of the heterotetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS·LZ PKG-Iα low-affinity heterotetrameric complex and allow reevaluation of the role(s) of myosin light-chain phosphatase partner polypeptides in regulation of vascular smooth-muscle cell contractility.

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“…13 C/ 15 N-or 15 N-labeled CC MBS polypeptides of Ͼ96% apparent purity migrated as a single homodimeric peak on SEC (Fig. 1B), consistent with previous results (16). Fig.…”

Section: Protein Purification and Nmr Spectroscopysupporting

confidence: 91%

mentioning

confidence: 52%

NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function

Sharma

Birrane

Anklin

et al. 2017

Journal of Biological Chemistry

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“…MALDI-TOF-determined molecular masses of N-terminally His 6 -tagged proteins may vary due to loss of the initiator Met [12,13]. The experimental mass of each evaluated APOL1 CC polypeptide indeed reflected loss of one Met, as shown in Fig.…”

Section: Maldi-tof Msmentioning

confidence: 76%

“…Buffer‐corrected spectra were recorded for 18 μ m APOL1 CC domain. Data were converted into MRE (θ x10 −3 ) (deg cm 2 ·dmol −1 ) . Thermal stability of CC variants was measured by CD thermal denaturation experiments conducted at 220 nm (ellipticity minima) by heating the samples from 25 °C to 95 °C at a rate of 50 °C·h −1 , with 3‐min thermal equilibration periods.…”

Section: Methodsmentioning

confidence: 99%

“…Isotopic labeling was therefore limited to G0 and G1 constructs. Uniformly 15 N-or 13 C/ 15 N-enriched APOL1 G0 and G1 CC polypeptides were expressed in M9 minimal medium containing 15 NH 4 Cl (1 gÁL À1 ) and 13 C glucose (2 gÁL À1 ) as sole nitrogen and carbon sources, and 10% of uniformly 15 N-or 13 C/ 15 N-labeled Bioexpress cell growth media (Cambridge Isotopes Laboratories, Tewksbury, MA, USA), in a manner identical to that used for their unlabeled counterparts. Clarified supernatants were equilibrated with buffer A and loaded at 4°C onto a Ni-nitrilotriacetic acid-Sepharose column (Amersham) pre-equilibrated with buffer A containing 1% Triton X-100, using a peristaltic Econo-pump (Bio-Rad, Hercules, CA, USA).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

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Structural characterization of the C‐terminal coiled‐coil domains of wild‐type and kidney disease‐associated mutants of apolipoprotein L1

et al. 2016

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Trypanosomes that cause sleeping sickness endocytose Apolipoprotein L1 (APOL1)-containing Trypanolytic Factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome T. brucei rhodesiense counteracts trypanolysis by expressing the surface protein SRA, which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C-terminal predicted coiled-coil (CC) domain of human APOL1, G1 (S342G/I384M) and G2 (ΔN388Y389), decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and MD simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with common variant G0. Conformational plasticity in G1 and G2 CC domains led to inter-helical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered structure of both helix α2 and nearby helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease.

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Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies

Cited by 2 publications

References 41 publications

NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function

NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function

Structural characterization of the C‐terminal coiled‐coil domains of wild‐type and kidney disease‐associated mutants of apolipoprotein L1

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