Figure 2. Intersubunit crosstalk maintains singly GTP-loaded state of the Rag GTPase heterodimer.
(A) Experimental setup for a sequential binding assay to probe the effect of xanthine nucleotide-loaded RagA on guanine nucleotide binding to RagC. Rag mutants with modified hydrogen-bonding pattern decouple nucleotide binding to the subunits.
(B) XTP loading on RagA(D130N) inhibits GTP binding to RagC. Top panel: GTP binding to RagC when RagA(D130N) is pre-bound to XTP or XDP. Bottom panel: quantification. When RagA(D130N) binds XTP, RagC loads a significantly smaller amount of GTP than when it binds XDP.
(C) Experimental setup of a sequential chase assay to probe the effect of later-bound nucleotides on the first-bound nucleotide.
(D) Later-bound XTP to RagA(D130N) has no effect on the amount of GTP pre-bound to RagC. Top panel: Pre-bound GTP to RagC chased by XTP or XDP binding on RagA(D130N). Bottom panel: quantification. Later-bound XTP or XDP on RagA(D130N) has no effect on the pre-bound GTP.
(E) Experimental setup of a half-site binding assay to measure the on-rates of nucleotides to singly-loaded Rag GTPases. Unlabeled nucleotides were added at sub-equal molar amounts compared to the nucleotide-binding pockets to generate singly-loaded Rag GTPases.
(F) Half-site on-rate measurements for GTP association when the Rags are singly-loaded with GTP (filled circle) or GDP (open circle). Subunit in bold is the subunit associated with the labeled GTP in the assay to measure the half-site on-rate. For example, RagA-RagCGDP (open red circle) shows how fast RagA associates with labeled GTP when RagC is preloaded with GDP. The slope gives the half-site on-rates. Notice that concentration-dependence is lost in the case of GTP.
(G) Half-site on-rate measurements for GDP association when the Rags are singly-loaded with GTP (filled circle) or GDP (open circle). As before, subunits in bold indicates which subunit is binding the labeled GDP in the assay to measure the half-site on-rate. GDP associates with the Rag GTPases more slowly if one subunit is preloaded with GTP.
(H) Summary of half-site on-rates for wildtype Rag GTPases at 4°C. Grey numbers in parenthesis denote the standard deviations of the reported values calculated from at least three independent experiments.
(I) Experimental setup for measuring the on-rates of nucleotides to the guanine-specific subunit when the xanthine-specific subunit is preloaded.
(J) On-rate of GTP association to RagC when RagA(D130N) was preloaded with XTP (filled circle) or XDP (open circle).
(K) On-rate of GTP association to RagA when RagC(D181N) was preloaded with XTP (filled circle) or XDP (open circle).
(L) Summary of differential on-rates for GTP at 4 °C. Grey numbers in parenthesis denote the standard deviations of the reported values calculated from at least three independent experiments.
(M) Illustration of the locked conformation induced by the binding of the first GTP to RagA and its inhibition of the association of the second GTP with RagC. The reverse case is shown in Supplementary Figure 2Q.