TY - JOUR
T1 - Structural insights into plasmalemma vesicle-associated protein (PLVAP)
T2 - Implications for vascular endothelial diaphragms and fenestrae
AU - Chang, Tao Hsin
AU - Hsieh, Fu Lien
AU - Gu, Xiaowu
AU - Smallwood, Philip M.
AU - Kavran, Jennifer M.
AU - Gabelli, Sandra B.
AU - Nathans, Jeremy
N1 - Funding Information:
We thank Dr. Eugene Butcher (Stanford University) for sharing the MECA-32 hybridoma cell line; John Williams for constructing several plasmids shown in Fig. 3; the Phasing@Home organizers Dr. Claudia Millán, Dr. Massimo Sammito, and Dr. Isabel Usón at Institut de Biologia Molecular de Barcelona for helpful discussions about the phase problem; Dr. Randy Read (Cambridge University) and Dr. Thomas Terwilliger (Los Alamos National Laboratory) for helpful discussions about MR-SAD; Dr. Jean Jakoncic and Dr. Alexei Soares for excellent support at the AMX beamline; and Dr. Yun- Sil Lee for helpful comments on the manuscript. This study was supported by the National Eye Institute (NIH) R01EY018637 (J.N.), the HHMI (J.N., T.-H.C., F-L.H, and P.M.S.), a Human Frontiers Postdoctoral Fellowship LT000130/2017-L (T.-H.C), the National Cancer Institute (NIH) (R01CA204345) (S.B.G.), and the National Institute of General Medical Sciences (NIH) R01GM134000 (J.M.K.). Work at the AMX (17-ID-1) and FMX (17-ID-2) beamlines was supported by the NIH, the National Institute of General Medical Sciences (P41GM111244), the US Department of Energy (DOE) Office of Biological and Environmental Research (KP1605010), and the National Synchrotron Light Source II at the Brookhaven National Laboratory, which is supported by the DOE Office of Basic Energy Sciences under contract DE-SC0012704 (KC0401040).
Funding Information:
ACKNOWLEDGMENTS. We thank Dr. Eugene Butcher (Stanford University) for sharing the MECA-32 hybridoma cell line; John Williams for constructing several plasmids shown in Fig. 3; the Phasing@Home organizers Dr. Claudia Millán, Dr. Massimo Sammito, and Dr. Isabel Usón at Institut de Biologia Molecular de Barcelona for helpful discussions about the phase problem; Dr. Randy Read (Cambridge University) and Dr. Thomas Terwilliger (Los Alamos National Laboratory) for helpful discussions about MR–SAD; Dr. Jean Jakoncic and Dr. Alexei Soares for excellent support at the AMX beamline; and Dr. Yun-Sil Lee for helpful comments on the manuscript. This study was supported by the National Eye Institute (NIH) R01EY018637 (J.N.), the HHMI (J.N., T.-H.C., F-L.H, and P.M.S.), a Human Frontiers Postdoctoral Fellowship LT000130/2017-L (T.-H.C), the National Cancer Institute (NIH) (R01CA204345) (S.B.G.), and the National Institute of General Medical Sciences (NIH) R01GM134000 (J.M.K.). Work at the AMX (17-ID-1) and FMX (17-ID-2) beamlines was supported by the NIH, the National Institute of General Medical Sciences (P41GM111244), the US Department of Energy (DOE) Office of Biological and Environmental Research (KP1605010), and the National Synchrotron Light Source II at the Brookhaven National Laboratory, which is supported by the DOE Office of Basic Energy Sciences under contract DE-SC0012704 (KC0401040).
Publisher Copyright:
Copyright © 2023 the Author(s).
PY - 2023/4/4
Y1 - 2023/4/4
N2 - In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ∼2/3 of the ∼390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.
AB - In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ∼2/3 of the ∼390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.
KW - alpha helix
KW - coiled-coil
KW - permeability
KW - single-wavelength anomalous dispersion of sulfur atoms
KW - vasculature
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U2 - 10.1073/pnas.2221103120
DO - 10.1073/pnas.2221103120
M3 - Article
C2 - 36996108
AN - SCOPUS:85151573831
SN - 0027-8424
VL - 120
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 14
M1 - e2221103120
ER -