Cooperative energetic effects elicited by the yeast Shwachman-Diamond syndrome protein (Sdo1) and guanine nucleotides modulate the complex conformational landscape of the elongation factor-like 1 (Efl1) GTPase.

One of the final maturation steps of the large ribosomal subunit requires the joint action of the elongation factor-like 1 (human EFL1, yeast Efl1) GTPase and the Shwachman-Diamond syndrome protein (human SBDS, yeast Sdo1) to release the eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) and allow the assembly of mature ribosomes. EFL1 function is driven by conformational changes. However, the nature of such conformational changes or the mechanism by which they are prompted are still largely unknown. In previous studies, it has been established that this GTPase interacts with its cofactor in solution in an inverted orientation with respect to the binding mode derived from 60S ribosome subunit cryo-EM data. To shed new light on this conundrum, we characterized calorimetrically the energetic basis describing the recognition of Efl1 to GT(D)P, Sdo1 and their intercommunication in solution. A structural-based analysis of the binding signatures indicates that Efl1 has a large structural flexibility. The mutual effects of Sdo1 and nucleotides on Efl1 modulate in a very specific and robust way the complex conformational landscape of Efl1, resembling the behavior observed with other GTPases and their cofactors.

Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation.

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.

Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.