Cryo-EM structure of the photosynthetic RC-LH1-PufX supercomplex at 2.8-angstrom resolution

The reaction center (RC)−light-harvesting complex 1 (LH1) supercomplex plays a pivotal role in bacterial photosynthesis. Many RC-LH1 complexes integrate an additional protein PufX that is key for bacterial growth and photosynthetic competence. Here, we present a cryo–electron microscopy structure of the RC-LH1-PufX supercomplex from Rhodobacter veldkampii at 2.8-Å resolution. The RC-LH1-PufX monomer contains an LH ring of 15 αβ-polypeptides with a 30-Å gap formed by PufX. PufX acts as a molecular “cross brace” to reinforce the RC-LH1 structure. The unusual PufX-mediated large opening in the LH1 ring and defined arrangement of proteins and cofactors provide the molecular basis for the assembly of a robust RC-LH1-PufX supercomplex and efficient quinone transport and electron transfer. These architectural features represent the natural strategies for anoxygenic photosynthesis and environmental adaptation

A human endothelial cell-based recycling assay for screening of FcRn targeted molecules.

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation. Nat Commun 2018 Feb 12; 9(1):621

Structural basis for the assembly and quinone transport mechanisms of the dimeric photosynthetic RC-LH1 supercomplex

The reaction center (RC) and light-harvesting complex 1 (LH1) form a RC–LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple phototrophic bacteria. Some species possess the dimeric RC–LH1 complex with a transmembrane polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC–LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC–LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC–LH1 dimer, interlocking association between the components and mediating RC–LH1 dimerization. Moreover, we identify another transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations provide a mechanistic understanding of the assembly and electron transport pathways of the RC–LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex

Structural basis for the assembly and electron transport mechanisms of the dimeric photosynthetic RC–LH1 supercomplex

AbstractThe reaction center (RC) and light-harvesting complex 1 (LH1) form a RC–LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple photosynthetic bacteria. Some species possess the dimeric RC–LH1 complex with an additional polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC–LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC–LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC–LH1 dimer, interlocking association between the components and mediating RC–LH1 dimerization. Moreover, we identify a new transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations enable a mechanistic understanding of the assembly and electron transport pathways of the RC–LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex.</jats:p

Unravelling the Roles of Integral Polypeptides in Excitation Energy Transfer of Photosynthetic RC-LH1 Supercomplexes.

Elucidating the photosynthetic processes that occur within the reaction center-light-harvesting 1 (RC-LH1) supercomplexes from purple bacteria is crucial for uncovering the assembly and functional mechanisms of natural photosynthetic systems and underpinning the development of artificial photosynthesis. Here, we examined excitation energy transfer of various RC-LH1 supercomplexes of Rhodobacter sphaeroides using transient absorption spectroscopy, coupled with lifetime density analysis, and studied the roles of the integral transmembrane polypeptides, PufX and PufY, in energy transfer within the RC-LH1 core complex. Our results show that the absence of PufX increases both the LH1 → RC excitation energy transfer lifetime and distribution due to the role of PufX in defining the interaction and orientation of the RC within the LH1 ring. While the absence of PufY leads to the conformational shift of several LH1 subunits toward the RC, it does not result in a marked change in the excitation energy transfer lifetime

An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers

An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers