Gao Pu and Zhang Liguo and collaborators revealed the transmembrane transport mechanism of cyclic dinucleotide and folic acid

Cyclic dinucleotides (CDNs) are widespread and important messenger molecules
in nature.
Mammalian cells produce a specific CDN:2'3'-cGAMP, which is catalyzed by the natural immune receptor cGAS after sensing abnormal DNA signals in the cytoplasm
.
2'3'-cGAMP binds to and activates the downstream linker protein STING, which in turn induces a broad-spectrum immune response
.
In addition to 2'3'-cGAMP, a variety of CDNs secreted by pathogenic bacteria and CDN anticancer drugs of wide concern can activate STING and regulate immune responses
.

One of the questions that has been a constant concern in the field is how polar CDN molecules enter cells across membranes.
Studies have shown that the solute carrier family protein SLC19A1 is a key CDN transporter
.
In addition to CDN, SLC19A1 is a major folate and antifolate transporter
.
Interestingly, some antifolate drugs are often used to treat some autoimmune diseases
through some "magical and unknown mechanism".
SLC19A1 is speculated to play a role
in this process due to its ability to transport these two types of substrates at the same time and the mutual inhibition of the transport of these two types of substrates.

In view of the important functions of SLC19A1 in CDN, folic acid and anti-folate transmembrane transport, the study of its substrate recognition mechanism will contribute to the mechanistic analysis of SLC19A1-related diseases and the development and optimization
of potential drugs.
On October 20, 2022, the Gao Pu team, Zhang Liguo's team from the Institute of Biophysics of the Chinese Academy of Sciences and the Gao team from Beijing Institute of Technology published an article "Recognition of cyclic dinucleotides and folates by human SLC19A1" online in the journal Nature, unveiling the mystery of SLC19A1's identification of different substrate molecules
。

The researchers first performed extensive screening and obtained a series of murine monoclonal antibodies that recognize SLC19A1 to stabilize protein samples
.
Using one of the rigidly binding antibodies, the researchers then resolved the substrate-free state electron microscopy structure
of SLC19A1 that opens inward-open to the inside of the cell.
SLC19A1 is an MFS-type transporter composed of 12 transmembrane helices, which identify key elements and intramolecular interactions in SLC19A1 inward-open conformation
.
The lumen of the SLC19A1 substrate channel and the cytosolic side inlet carry a large amount of positive charge, which is consistent with the anionic properties of the substrate transported by SLC19A1
.
By mapping reported deficits of function or disease-associated mutations, the researchers found that most of these mutations affected substrate recognition, transport, or the protein's own folding
.

Next, the researchers resolved the electron microscopy structure
of SLC19A1 in the inward-open state of three CDN complexes from different sources.
Surprisingly, all three CDN molecules bind to the bottom of the positively charged cavity of SLC19A1 by forming compact and delicate dimer units, a substrate recognition that has not been reported
in other SLC or MFS family proteins.
The CDN dimer unit presents a conformation of bases in the middle and phosphate glycan rings at both ends, and this particular assembly method relies on the stacking and hydrogen bonding interaction between the two CDN molecules
.
Although the binding patterns of different CDNs are roughly similar, there are obvious conformational differences in their respective dimer units, which reflects the broad inclusiveness
of SLC19A1 for different CDNs.

To explore the mechanism by which SLC19A1 recognizes folic acid and antifolate, the researchers successfully resolved the high-resolution electron microscopy structure
of SLC19A1 with 5-MTHF (reduced folic acid mainly present in diet and blood) and PMX (a new class of antifolic acid drugs) complexes using a new set of antibodies.
However, unlike CDN, both 5-MTHF and PMX are monomerically bound to the middle and upper part
of the SLC19A1 polar cavity.
SLC19A1 was previously known to transport reduced folate much more efficiently than regular folate, and the researchers found that SLC19A1 formed an additional interaction
with 5-MTHF methyl group and 8-position hydrogen atoms.

Although CDN and folate/antifolic acid bind sites to SLC19A1 are completely different, both substrates are transported through the same internal channel, so there is clearly competitive inhibition
between the two.
The researchers conducted a systematic mutation analysis of the two substrate binding pockets and found that some mutations affect the transport of both types of substrates at the same time, while others only have a significant impact on the transport of one type of substrate, suggesting that it is possible to design small molecule drugs
with substrate inhibitory selectivity.

SLC19A1 transport CDN (a) and folic acid/anti-folic acid (b) concept diagram and model diagram

In summary, this study reports the molecular basis of human SLC19A1 to recognize different substrates such as CDN, folic acid and anti-folic acid, reveals its unique and diverse substrate recognition mechanism, and identifies a new substrate recognition mode
of SLC and MFS families.
This study provides a new idea for the development of a new generation of CDN drugs and anti-folic acid drugs, and the antibody screening platform established in the study and the obtained monoclonal antibodies also provide a basis
for the development of antibody drugs.

Zhang Qixiang, doctoral student of the Institute of Biophysics, Xuyuan Zhang, research assistant of the Institute of Biophysics, Yalan Zhu, postdoctoral fellow of Beijing Institute of Technology, and Panpan Sun, doctoral student of the Institute of Biophysics, are the co-first authors of this paper.
Prof.
Pu Gao, Prof.
Liguo Zhang and Prof.
Gao are co-corresponding authors
.
The research team of Lou Jizhong of the Institute of Biophysics provided core support in computational simulation, the Wang Feng team provided assistance in antibody screening, and the Liu Zhenfeng research team provided assistance
in the preparation of membrane proteins.
The research was supported
by the National Natural Science Foundation of China, the Ministry of Science and Technology, the Beijing Municipal Natural Science Foundation of China, and the Chinese Academy of Sciences.
Relevant experiments are supported and helped
by the protein science research platform of the Institute of Biophysics.

Article link: https://doi.
org/10.
1038/s41586-022-05452-z

(Contributed by: Gao Pu Research Group)