Protein interactions are at the heart of a cell's function. Calorimetric and spectroscopic techniques are commonly used to...

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond Syndrome, release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a Guanine nucleotide Exchange Factor (GEF) for EFL1 promoting its activation and the release of GDP. Finally, fluorescence anisotropy measurements showed that the mutation S143L present in the Shwachman-Diamond Syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1.

The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in the genes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPase known as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-association factor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of mature ribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes. The molecular alterations resulting from the latter remain largely unknown, although some evidence suggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutations on the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1. Binding was either severely weakened or almost abolished, depending on the assessed mutation. In higher eukaryotes, SBDS is essential for development, and lack of the protein results in early lethality. The existence of patients whose only source of SBDS consists of that with surface missense mutations highlights the importance of the interaction with EFL1 for their function. Additionally, we studied the interaction mechanism of the proteins in solution and demonstrated that binding consists of two independent and cooperative events, with domains 2–3 of SBDS directing the initial interaction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited large flexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-ray Scattering (SAXS) experiments.