Proteins are linear chains of amino acids that fold into complex three-dimensional structures associated with particular functions. Extensive structural analyses of proteins by X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy have revealed that many proteins are modular, and consist of domains that fold independently. If an amino acid sequence folds into a particular structure, it is a safe bet that a similar sequence in a second protein will adopt a similar structure. Hundreds of unique modules have been defined and archived in databases that allow prediction of structure and function on the basis of amino acid sequence. However, sequences still exist for which structure cannot be predicted. Such was the case for myeloid-derived growth factor (MYDGF), a protein of ~145 amino acids present in nearly every tissue and cell in the human body and in organisms as distant from humans as slime molds.
In a major step forward in understanding MYDGF, a recent Nature Communications paper out of Deane Mosher’s lab describes the three-dimensional structure of human MYDGF solved in collaboration with John Markley and the National Magnetic Resonance Facility at Madison (NMRFAM). First author Valeriu Bortnov is a PhD student in the Integrated Program in Biochemistry. In an earlier paper in the Journal of Biological Chemistry, Bortnov showed that MYDGF localizes to the endoplasmic reticulum (ER) and Golgi, the organelles of the cell responsible for trafficking of proteins to outside of the cell. Both ER proteins and trafficked proteins move from ER to Golgi, but whereas trafficked proteins enter vesicles that continue to the outside, ER proteins bind to so-called ‘KDEL receptors’ and are retrieved back to the ER. Mosher likens such protein trafficking to a distillation. For MYDGF, however, the distillation back to the ER appears to break down under certain circumstances, because others had shown that MYDGF is released from cells and mediates tissue repair after heart attacks in mice. Bortnov explains, “While identifying a potential role for MYDGF in the treatment of heart attacks is a very intriguing finding, our working hypothesis is that the secretion of MYDGF from the cell is highly dependent on the local environment, in particular on circumstances where cells are highly stressed, such as heart attacks. Even though MYDGF is a resident ER protein under standard physiological conditions, it could also act as a beneficial, secreted growth factor for neighboring cells during instances of cell stress.”
“Knowledge of the structure of MYDGF was needed to solidify our thinking and go further,” Bortnov adds. To tackle the structure of MYDGF by NMR spectroscopy required making protein in bacteria grown on the NMR-active isotopes 15N and 13C, and this meant that the UW-Madison team first had to show that MYDGF produced by bacteria folded into the same conformation as the MYDGF studied earlier, which was produced in and secreted through the ER of eukaryotic insect cells. The two preparations of MYDGF were found to be identical by ‘top-down’ mass spectrometry done in collaboration with Ying Ge of UW-Madison’s Human Proteomics Program and by low-resolution biophysical measurements carried out in the campus’ Biophysics Instrumentation Facility. With assurance that the protein was properly folded, Bortnov and NMRFAM collaborators determined the structure of MYDGF in solution under closely physiological conditions. Fortuitously, a crystal structure of a KDEL receptor was published in early 2019, allowing computational docking of MYDGF onto the receptor, carried out in collaboration with Julie Mitchell, formerly at UW-Madison and now at Oak Ridge National Laboratory. Finally, a comparison of the MYDGF structure with known structures revealed that the base domain of an enzyme called ‘vanin’ adopts the same fold as MYDGF, and this finding led collaborator Alex Batemen of the European Molecular Biology Laboratory/European Bioinformatics Institute to create a new protein family (Pfam) encompassing sequences related to those of the MYDGF and vanin base modules.
The summary figure below created by Bortnov shows the structure of MYDGF (blue) docked onto a KDEL receptor (purple). The expanded image of MYDGF on the right highlights conserved amino acid residues (dark blue) and a potential pocket (red) that may bind small molecules. Bortnov explains, “The two residues at the tail are required for binding to the KDEL receptor, which stabilizes its retention in the ER, and the conserved residues in the opposite region are also likely critical for function. This region is a possible site for interactions with other ER proteins or small molecules and is accessible even when MYDGF is bound to the KDEL receptor.” A separate paper published almost simultaneously in Nature Communications on the X-ray crystal structure of MYDGF found nearly the same protein fold and concluded that this same face of MYDGF is important for its reparative function in the heart.
The summary figure to the left, created by Bortnov, shows the structure of MYDGF (blue) docked onto a KDEL receptor (purple). The expanded image of MYDGF on the right highlights conserved amino acid residues (dark blue) and a potential pocket (red) that may bind small molecules. Bortnov explains, “The two residues at the tail are required for binding to the KDEL receptor, which stabilizes its retention in the ER, and the conserved residues in the opposite region are also likely critical for function. This region is a possible site for interactions with other ER proteins or small molecules and is accessible even when MYDGF is bound to the KDEL receptor.” A separate paper published almost simultaneously in Nature Communications on the X-ray crystal structure of MYDGF found nearly the same protein fold and concluded that this same face of MYDGF is important for its reparative function in the heart.
Now that they have a structure to work with, Bortnov and others at UW-Madison are trying to understand more about what MYDGF does. While other teams are more focused on what happens when it is secreted, the protein likely has more evolutionarily-conserved functions within the cell. “For nearly every species all the way to single-cell eukaryotes, the MYDGF protein contains an ER retention tag. So presumably in addition to acting as a secreted reparative tissue factor, it also has conserved functions in the ER,” says Bortnov. “We now want to know what proteins or molecules it binds to and under what conditions. If it does have a dual role, what is it doing in the ER physiologically?”
Finding answers to these questions could help guide researchers toward solidifying potential therapeutic applications for MYDGF. You can read the full paper, Solution structure of human myeloid-derived growth factor suggests a conserved function in the endoplasmic reticulum, in Nature Communications here.