A pathway emerges: Biologists describe structure and function of a heme transport and assembly machine

Date:
December 20, 2021
Source:
Washington University in St. Louis
Summary:
Researchers described for the first time the structure of a
bifunctional protein, called CcsBA, that transports heme and
attaches it to cytochromes. The study captured two conformational
states of CcsBA, a bacterial and chloroplast protein, allowing
scientists to characterize the enzyme mechanism.



FULL STORY ==========================================================================
Heme is an essential part of the protein hemoglobin, which colors human
blood red. Heme also is crucial for cytochrome proteins, which power
the cell.

Humans, animals, plants and bacteria all use heme.


========================================================================== Hemoglobin shuttles oxygen to tissues where it is needed, while
cytochromes carry electrons for energy conversion in the cell. But understanding how heme moves across membranes -- like it needs
to, in order to insert into hemoglobin and cytochromes -- has been
challenging. Heme transport is transient, which means heme moves through membranes quickly and leaves behind no traces. And heme-binding membrane proteins are difficult to purify in large quantities.

In research published Dec. 20 in Nature Chemical Biology, scientists
at Washington University in St. Louis described for the first time the structure of a bifunctional protein, called CcsBA, that transports
heme and attaches it to cytochromes. The study led by Robert Kranz,
professor of biology in Arts & Sciences, captured two conformational
states of CcsBA, a bacterial and chloroplast protein, allowing scientists
to characterize the enzyme mechanism.

"This new paper addresses the structural basis for how the CcsBA machine functions, revealing major dynamic switches that occur during the cycle
of heme transport," Kranz said.

The study was made possible by a collaboration with James Fitzpatrick,
director of the Washington University Center for Cellular Imaging
(WUCCI) at the School of Medicine and a professor of neuroscience, of
cell biology and physiology, and of biomedical engineering, and Michael
Rau, a staff scientist and structural biologist on his team. They
leveraged a cutting-edge structural biology technique called single
particle averaging, which utilized a state-of- the-art cryo-Electron
Microscope (cryo-EM) to image different views of the protein in its
natively vitrified (frozen) state. After sorting all of the different
views, they were able to construct cryo-EM density maps -- which are three-dimensional representations of the protein built from a series of
two- dimensional projections of various views -- from which Kranz's team
could build an atomic model of the structure of CcsBA.

"Cryo-EM is a transformative technology that allows us to visualize at
the near-atomic level the structural arrangement of a given protein,
including the ability to tease out different conformations from an
ensemble of states," Fitzpatrick said. "It was this latter ability that
was key in enabling us to capture the mechanism of heme transport."
The cyro-EM data identified two states in which either one or two heme molecules were bound.



==========================================================================
"The structural models we were able to construct illustrate that CcsBA
is trapped with heme in two different conformations, which we term the
closed and open states," Kranz said. "This new body of work addresses
the structural basis by which the CcsBA machine functions, revealing a
major dynamic switch that occurs during the transport cycle.

"One of the coolest findings is that a large chamber opens upon heme transport," he said. The chamber is for cytochrome c synthesis.

Co-first author Deanna L. Mendez, a postdoctoral staff scientist
in biology, previously co-authored a study with Kranz in eLIFE
about the reconstitution of the purified bacterial and human
synthases of heme. CcsBA is different from the human form of heme-transporter/cytochrome c synthase. The insights gained through
identifying its structures give researchers a leg up on developing antimicrobial agents that will selectively target bacteria.

"In CcsBA, we observe a clear path between transmembrane alpha helices
that could allow heme to travel from the transmembrane-heme site to the external heme site," Mendez said. "Since heme goes down its concentration gradient during export, we do not envision an energy source requirement
for this process." Co-first author Ethan Lowder is a senior at Washington University and worked on this project for two years.



========================================================================== "Solving a novel structure of a protein is very difficult," Lowder
said. "In this case, there were no similar structures that we could rely
on as a template or starting point. It was definitely a challenge, but we worked all the way through it to get to the final structures." The second author, Dustin Tillman, who was an undergraduate at Washington University
when he completed this work, was instrumental in purifying CcsBA. He
partnered with the WUCCI team to optimize the sample preparation for the
single particle cryo-EM studies. "Dustin also performed reconstitution
assays on his purified preps to show they were active synthases,"
Kranz said.

"I am grateful for the contributions and involvement of our talented undergraduate researchers in this NIH-supported research," Kranz
said. "This study is a culmination of over three decades that our
lab has studied heme transport and cytochrome assembly, so it is
satisfying to know the structural basis for both. It will lead to
more experiments on mechanisms of chamber opening, transport and the
synthase reaction in the chamber." Video: https://youtu.be/lX1oN9XNxXQ ========================================================================== Story Source: Materials provided by
Washington_University_in_St._Louis. Original written by Talia
Ogliore. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Deanna L. Mendez, Ethan P. Lowder, Dustin E. Tillman, Molly C.

Sutherland, Andrea L. Collier, Michael J. Rau, James
A. J. Fitzpatrick, Robert G. Kranz. Cryo-EM of CcsBA reveals
the basis for cytochrome c biogenesis and heme transport. Nature
Chemical Biology, 2021; DOI: 10.1038/s41589-021-00935-y ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/12/211220120055.htm

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