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On January 3, 2022, Professor Gao Ning's research group from the School of Life Sciences, Peking University published a research paper entitled " Structural insights into the membrane microdomain organization by SPFH family proteins" in Cell Research , using cryo-electron microscopy to study bacteria located in bacteria.
The high-resolution structure of the supermembrane complex formed by two representative SPFH family protein molecules (HflK, HflC) and the AAA+ protease FtsH in the inner membrane reveals that the SPFH family proteins have different intracellular membrane system microdomains (microdomains).
) common principles and molecular basis in organizational processes .
The composition of the cell membrane system and the distribution of lipids and proteins are all highly heterogeneous
.
A common feature of the cell membrane systems of prokaryotes and eukaryotes is that they all have relatively discrete functional membrane microdomains (FMMs, functional membrane microdomains), but the molecular organization basis of such micromembrane domains has been unknown
The SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) family is a class of membrane proteins localized to a variety of biological membrane systems, including plasma membrane, nuclear membrane, Golgi apparatus, endoplasmic reticulum, endosome, mitochondria, chloroplast, even lipid droplets (Browman et al.
, 2007)
.
SPFH family proteins are hallmark molecules in FMM, and based on their oligomeric properties, it is speculated that they may play a basic scaffolding role in the formation and organization of FMM
Although the SPFH family is numerous and widespread in all species and participates in many basic biological processes, the oligomeric organization, three-dimensional structure of SPFH family proteins and their mechanisms for regulating various membrane-associated molecular processes are very unclear
.
In this study, KCF was used as a model complex of the SPFH family, and the composition and high-resolution three-dimensional structure of the KCF complex was studied by the three-dimensional reconstruction technique of cryo-electron microscopy
First, the cryo-electron microscopy data showed that HflK/C was a heterodimer as the basic unit, and 12 HflK molecules and 12 HflC molecules were spaced on the inner membrane to form a cage-like structure of transmembrane heterotetramers
.
The N-terminal region of HflK/C forms and isolates a circular membrane region with a diameter of approximately 20 nm; most of the amino acid residues of HflK/C are located in the periplasmic space and assemble into a completely sealed cage like structure
Figure 1: Overall structural overview of KCF complexes containing different numbers of FtsH hexamers
Second, based on the analysis of the high-resolution structure (3.
3 Å) of the KCF complex containing 4 FtsH hexamers, the paper found some previously unknown shared structural properties of the SPFH family (Fig.
2): SPFH family-specific SPFH domains Can be precisely divided into two domains (SPFH1 and SPFH2), of which the SPFH1 domain is actually an intercalated domain, approximately half of which is inserted into the outer leaflet of the cell membrane; the adjacent SPFH1 structure The domains interact closely with each other, demarcating a 20 nm diameter microdomain completely separate from the surrounding lipids directly on the outer lobe
.
The high-resolution structures clearly demonstrate the intact oligomeric forms of HflK and HflC interacting side-by-side from the N- to C-termini: in addition to the interacting SPFH1 and SPFH2 domains, the intermediate domains (single alpha helix, CC1 and CC2) also interact closely with each other, forming the walls of the cage through right-handed helices; the domain closest to the C-terminus contains a characteristic β-strand (β9), which is contributed by all HflK and HflC subunits in the complex, respectively A β-strand, thus forming a stable β-barrel
Figure 2: Structural model, domain composition and formation of membrane microdomains of HflK/C
The paper further determined the amino acid sequence of HflK subunit involved in recognizing and binding FtsH
.
Based on these structural information and biochemical experimental data, the molecular mechanism of HflK and HflC regulating bacterial membrane protein homeostasis through FtsH is proposed: Under normal physiological conditions, the HflK/C cage structure can spatially isolate FtsH protease, thereby preventing FtsH Degradation of functional membrane proteins on the cell membrane is essential for maintaining the stability of membrane proteins located on abnormally crowded cell membranes; for abnormally assembled membrane proteins, or damaged membrane protein complexes, the free ends of membrane proteins are not free.
More importantly, the paper carried out a sequence-based structural analysis of typical SPFH family proteins, and found that the domain organization of all SPFH family proteins is highly similar to HflK/C, consisting of N-terminal SPFH1 and SPFH2 domains, and middle SPFH1 and SPFH2 domains.
CC domain, and the C-terminal β-sheet domain
.
These analyses suggest that all SPFH family members are likely to form cage-like structures in a similar manner at different subcellularly localized cell membrane systems, resulting in the formation of micromembrane domains of different sizes
Figure 3: A membrane protein quality monitoring model of the KCF complex and the structural basis of SPFH family proteins in FMM tissue
In conclusion, this study elucidates the organization of a very novel membrane protein complex through the structural and functional elucidation of HflK and HflC of the bacterial SPFH family, which is important for understanding the role of its mitochondrial homolog, Prohibitins, in human-related diseases.
The molecular mechanism in is very critical
.
More importantly, this work provides a "concrete" physical basis for understanding the "fuzzy" cell biology concept of FMM or lipid rafts
Cell Research published a Research Highlight entitled "SPFHprotein cage – one ring to rule them all"
in the same journal .
The review article, written by Oliver Daumke and Gary R.
Lewin of the Max Delbrück Center for Molecular Medicine in Germany, pointed out the significance of this paper for understanding the general molecular organization of functional micromembrane domains
.
Gao Ning is the corresponding author of this paper
.
Ma Chengying and Wang Chengkun, postdoctoral fellows in Gao Ning's group, are the co-first authors of this paper
.
This research was supported by the National Natural Science Foundation of China, the Joint Center for Life Sciences, the State Key Laboratory of Membrane Biology, and the Qidong Industrial Innovation Fund of the Academy of Biological Sciences, as well as the cryo-electron microscopy platform, electron microscopy laboratory, high-performance computing center of Peking University, Technical support from the Instrument Center of the Academy of Biological Sciences and the National Protein Infrastructure (Peking University sub-platform)
.
Ma Chengying and Wang Chengkun were supported by the postdoctoral fellowship from the Joint Center for Life Sciences
.