TY - JOUR
T1 - Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator yiip
AU - Lopez-Redondo, Maria
AU - Fan, Shujie
AU - Koide, Akiko
AU - Koide, Shohei
AU - Beckstein, Oliver
AU - Stokes, David L.
N1 - Funding Information:
Funding for this work was provided by National Institutes of Health grant 1R01GM125081 (D.L. Stokes) and by Aarhus Institute for Advanced Studies which is supported by the European Union’s 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 754513 and the Aarhus University Research Foundation (D.L. Stokes). Additional EM was performed at the Pacific Northwest Center for Cryo-EM at Oregon Health Sciences University, which was supported by National Institutes of Health grant U24GM129547 and accessed through EMSL (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. MD simulations were performed using PSC Bridges at the Pittsburgh Supercomputing Center (allocation TG-MCB130177), a resource of the Extreme Science and Engineering Discovery Environment, which is supported by National Science Foundation grant number ACI-1548562. The authors declare no competing financial interests.
Funding Information:
Joseph A. Mindell served as editor. EM was performed at the Cryo-EM Core Facility at New York University Langone Health, with the assistance of William J. Rice and Bing Wang. The authors acknowledge Research Computing at Arizona State University for providing HPC and storage resources that have contributed to the research results reported within this paper, and thank M. Bonomi for assistance in im-plementing software for comparing MD simulations and cryo-EM maps. Funding for this work was provided by National Institutes of Health grant 1R01GM125081 (D.L. Stokes) and by Aarhus Institute for Advanced Studies which is supported by the European Union?s 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 754513 and the Aarhus University Research Foundation (D.L. Stokes). Additional EM was performed at the Pacific Northwest Center for Cryo-EM at Oregon Health Sciences University, which was supported by National Institutes of Health grant U24GM129547 and accessed through EMSL (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. MD simulations were performed using PSC Bridges at the Pittsburgh Supercomputing Center (allocation TG-MCB130177), a resource of the Extreme Science and Engineering Discovery Environment, which is supported by National Science Foundation grant number ACI-1548562. The authors declare no competing financial interests.
Publisher Copyright:
© 2021 Lopez-Redondo et al.
PY - 2021/8/2
Y1 - 2021/8/2
N2 - YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.
AB - YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.
KW - Biophysics
KW - Membrane transport
KW - Protein structure and dynamics
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U2 - 10.1085/jgp.202112873
DO - 10.1085/jgp.202112873
M3 - Article
C2 - 34254979
AN - SCOPUS:85111428771
VL - 153
JO - Journal of General Physiology
JF - Journal of General Physiology
SN - 0022-1295
IS - 8
M1 - e202112873
ER -