NMR characterization of the interaction between siderocalin NGAL and its cellular receptor

The aim of this project is to determine, mainly by nuclear magnetic resonance (NMR), the structural and dynamic properties of the siderocalin cellular receptor 24p3R and of its interaction with the siderocalin NGAL. This protein complex has the striking particularity to retain inherent disorder ("fuzzy" protein complex), which excludes the use of conventional structural analysis tools. Therefore, we will propose and NMR-based strategy for the characterization of "fuzzy" protein complexes in order to decipher the functional properties of the NGAL/24p3R system. Our results should lead to a better understanding of the normal and pathological functions of NGAL/24p3R and address the potentiality of siderocalins and their receptor as targets for therapeutic intervention. Siderocalins are small secreted proteins characterized by their ability to transport iron via catechol type chelators. The prototypical siderocalin is the human neutrophil gelatinase associated lipocalin (NGAL). The NGAL cellular receptor, 24p3R, triggers the endocytosis of NGAL that can subsequently bind or deliver iron through the endosomal pathway. The endocytosis of NGAL promotes different physiological responses, from tissue differentiation to apoptosis, depending on NGAL iron content. Additionally, a growing set of evidence suggests that NGAL/24p3R is involved in cancer progression and metastasis. So far, no general picture exists that satisfactorily explains the pleiotropic function of NGAL/24p3R. Furthermore, the molecular determinants of NGAL/24p3R physiology are still largely to be deciphered. Mainly, there is a clear and detrimental lack of biochemical and structural data about the NGAL/24p3R system. 24p3R consists of 11 transmembrane helices and several large, soluble and poorly structured segments. Our preliminary data show that the 100 residues extracellular N-terminal domain of 24p3R is an intrinsically disordered protein (IDP) that binds NGAL with a low micromolar affinity to form a "fuzzy" complex where NGAL retains its fold but the N-terminal domain of 24p3R exhibits significant disorder. In order to understand the functional properties of the NGAL/24p3R system we will first determine the conformational behavior of the N-terminal domain of 24p3R by solution state NMR. We will also characterize the properties of the fuzzy complex formed by the N- terminal domain and NGAL. As no methodological framework has been proposed yet to address fuzzy complexes, this last part will require the development of a specific NMR based strategy to properly characterize this emerging type of protein complex. Finally, the short and functional splicing variant of 24p3R (22 kDa) will be addressed by solution state NMR. We will determine the structural and functional properties of this short splicing variant as well as its binding properties towards NGAL. We will also characterize the conformational behavior of the soluble N-terminal domain in the context of the full-length protein embedded in a membrane mimetic. Taken together, these data will allow us to determine the structural and dynamic properties of the NGAL/24p3R system, which should lead to a better understanding of the normal and pathological functions of NGAL/24p3R and address the potentiality of siderocalins and their receptor as targets for therapeutic intervention. Additionally, we will provide a detailed description of a fuzzy protein complex and propose an NMR-based strategy for the characterization of this emerging type of complex.

The aim of this project was to determine, mainly by nuclear magnetic resonance (NMR), the structural and dynamic properties of the siderocalin cellular receptor 24p3R and of its interaction with the siderocalin NGAL. Siderocalins are small secreted proteins characterized by their ability to transport iron via catechol type chelators. The prototypical siderocalin is the human neutrophil gelatinase associated lipocalin (NGAL). The NGAL cellular receptor, 24p3R, triggers the endocytosis of NGAL that can subsequently bind or deliver iron through the endosomal pathway. The endocytosis of NGAL promotes different physiological responses, from tissue differentiation to apoptosis, depending on NGAL iron content. Additionally, a growing set of evidence suggests that NGAL/24p3R is involved in cancer progression and metastasis. 24p3R consists of 11 transmembrane helices and several large, soluble and poorly structured segments. During the course of this project we have demonstrated, using NMR in conjunction with mass spectrometry and ex vivo immunofluorescence, that the 100 residues extracellular N-terminal domain of 24p3R (24p3R-NTD) is an intrinsically disordered protein (IDP) that binds NGAL with a low micromolar affinity to form a fuzzy complex where NGAL retains its fold but the N-terminal domain of 24p3R exhibits significant disorder. This type of complex is rather unusual, even for IDPs that usually fold upon binding, and cannot be addressed by conventional structural biology techniques. We used state of the art liquid state NMR in conjunction with other biophysical methods in order to unravel the structural dynamics of this intriguing complex. Namely, we exploited the fast exchange between the free and bound form of 24p3R-NTD in presence of sub-stoichiometric amounts of NGAL (typically 1:0.1). We used 15N-CPMG experiments on this type of samples in order to extract information about the bound state of 24p3R-NTD. Using this original methodology, we could show that several parts of the receptor are involved in the interaction with NGAL (a rather unusual feature for an IDP), and that 24p3R-NTD assumes at least three distinct, but interconverting conformations in the complex.To sum up, we contributed to a better understanding of the biochemistry of NGAL and its cellular receptor, which should lead to a better understanding of the normal and pathological functions of NGAL/24p3R and address the potentiality of siderocalins and their receptor as targets for therapeutic intervention. We also provided an example of an atypical interaction between a folded protein and an IDP. We demonstrated that an elegant usage of diverse NMR methods in conjunction with other biophysical techniques (ITC, MS) allows achieving an in depth characterization of this type of complex.