Structure-function analysis of a prominent target in fibrosis: the C-terminal maturation complex of fibrillar collagens – Pcube_complex

Fibrillar collagens are the most abundant proteins in the human body and any defect in their biosynthesis or the disruption of the balance between their synthesis and degradation cause major pathological disorders. These include rare genetic diseases such as osteogenesis imperfecta and numerous debilitating and life-threatening conditions resulting from defective tissue repair. Among the latter, fibrosis is characterized by excessive or aberrant collagen deposition and is a leading cause of death worldwide. Typical triggering factors of fibrosis are cardiovascular diseases, chronic kidney disease, infections (e.g. COVID19, hepatitis B or C), immune diseases (e.g. scleroderma, rheumatoid arthritis) or toxic insults (e.g. alcohol, high-fat diet). One promising strategy to control collagen deposition in fibrosis is to target fibrillar collagen biosynthesis. In this project, we will focus on the rate-limiting step of collagen biosynthesis, the C-terminal proteolytic maturation of major fibrillar collagens I-III, and try to understand at the atomic level how the various partners of the maturation complex associate and work together.
The targeted complex is minimally composed of a fibrillar collagen precursor (or procollagen) and a procollagen C-proteinase (or PCP). The main PCPs are extracellular metalloproteinases called Bone Morphogenetic Protein-1 (BMP-1)/Tolloid-like proteinases (BTPs) which remove the C-terminal globular domain of procollagens (or C-propeptide) and trigger collagen fibrillogenesis. The PCP activity of BTPs can be stimulated, with different efficiencies, in the presence of two enhancing glycoproteins, PCPE-1 and -2, which are considered to be the most promising targets in fibrosis among complex partners thanks to their high specificity for collagen biosynthesis. Therefore, obtaining the structure of at least one collagen maturation complex formed by a Procollagen, a PCP and a PCPE (summarized here as “P3-complex”), at the highest possible resolution, is the main objective of the present project. It should allow us to (i) better understand the roles of individual protein domains in the proteolytic reaction, (ii) explain differences in activities between the two regulatory proteins PCPE-1 and -2 and better define their individual therapeutic potential, (iii) describe interaction surfaces in the P3-complex and determine if they could be targeted for anti-fibrotic drug development.
Although significant progress has been made recently to understand the function and mechanism of the P3-complex, the questions mentioned above have been impossible to address in the past because of the difficulties to use BTPs in crystallographic studies. The new possibility to obtain medium-to-high resolution structures of protein complexes above 100 kDa by cryo-electron microscopy (cryo-EM) with relatively modest amounts of protein opens very promising perspectives and this technique will be at the centre of our proposal. We plan to use a stepwise approach going from the analysis of the two-partner complexes for which structural data are presently missing (PCP/procollagen and PCP/PCPE) to the analysis of the three-partner complexes (PCP/procollagen/PCPE). In addition, we will also rely on a combination of biochemical, biophysical, modelling and mutagenesis studies to optimize protein production and obtain complementary information on the mechanism of action of the various protein partners.
To implement this ambitious project, we will take advantage of the numerous protein tools and assays previously developed by Partner 1 (LBTI, France) and of the strong expertise in cryo-EM data collection and processing of Partner 2 (STRUBI, UK), with the aim to fully elucidate the molecular mechanisms underlying collagen C-terminal maturation and, in the long run, to design novel anti-fibrotic strategies.