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  • SC 79 On the basis of the structure

    2021-12-07

    On the basis of the structure of the Rab35/ACAP2 complex, the authors identified Thr76 in switch II of Rab35 as one key specificity determinant (Lin et al., 2019). The crystal structure shows that the hydroxyl group of Thr76 forms a SC 79 with Asp756 in ACAP2, while the methyl group is a part of the hydrophobic core of the binding interface. These structure features are consistent with a previous study showing the importance of Thr76 in ACAP2 binding (Etoh and Fukuda, 2015). Interestingly, the residue corresponding to Thr76 in Rab35 is highly divergent in the Rab family. Even in some of the closely related Rabs such as Rab1, Rab8, Rab10, and Rab13, this residue is replaced by either serine or alanine. Remarkably, Lin et al. (2019) showed that mutating Thr76 in Rab35 to serine led to a 10-fold decrease of the binding affinity to ACAP2 as measured by isothermal titration calorimetry, providing a basis for the specific binding of ACAP2 to Rab35 but not Rab1, Rab8, Rab10, or Rab13 or other more distally related Rabs. These results demonstrate that a single methyl group can make a substantial contribution to the binding energy, leading to highly specific binding of ACAP2 to Rab35. The structure also identifies residues in ACAP2 important for binding to Rab35. Many of these residues are not conserved in ACAP1 or ACAP3, explaining the lack of binding to Rab35 by these ACAP paralogs despite their similar structures. In the structure of the Rab35/RUSC2 complex, Asp4 in Rab35 makes a charge-charge interaction with Lys998 in RUSC2 at the periphery of the binding interface. Asp4 is located in the so-called complementary determination region 1 that is known to be involved in specific interactions with effectors (Pylypenko et al., 2018). Consistent with the structure, Lin et al. (2019) showed that mutating Asp4 to alanine dramatically decreased the Rab35/RUCS2 interaction in pull-down assays, although the binding affinity was not measured with a quantitative method. Similar to Thr76, Asp4 in Rab35 is not conserved in other Rab proteins. These analyses therefore strongly suggest that Asp4 is a key specificity determinant in the Rab35/RUSC2 interaction. An additional interesting feature of the Rab35/RUSC2 complex is the involvement of an arginine residue in RUSC2 (Arg1015) in the interaction with GTP bound to Rab35. Most GAPs of GTPases provides an “arginine finger” to directly interact with the γ -phosphate group in GTP, which is essential for catalyzing GTP hydrolysis. Arg1015 in RUSC2, however, approaches GTP from a different orientation and interacts with GTP indirectly through two water molecules. Consequently, Arg1015 in RUSC2 does not promote GTP hydrolysis of Rab35 and is therefore called a “pseudo-arginine finger.” By structural comparisons, Lin et al (2019) found that Thr72 in Rab35 is important for the interaction with ACAP2 but is unlikely to be involved in the interactions with other effectors such as MICAL and ORCL (Lin et al., 2019). Similarly, Asp4 in Rab35, which is important for binding RUSC2, appears to be unnecessary for the interactions with MICAL or ORCL. Indeed, the individual T72A and D4A mutations of Rab35 selectively disrupt the binding to ACAP2 and RUSC2, respectively, but have no negative effect on the interactions with MICAL or ORCL. These results suggest that carefully designed mutations of the specificity determinants can disrupt the interactions of Rab35 (or other small GTPases) with one effector but leave other interactions unperturbed, which can serve as useful tools for functional investigations of the particular interactions inside the cell.
    Introduction CgtA (Caulobactor GTPase A) is a ribosome associated GTPase protein that is essential for the survival of many bacteria. CgtA is a universally conserved GTPase, belonging to Obg class of GTPase. Expression of CgtA has been observed in several human pathogenic bacteria and it is being considered as a potential drug target [1,2]. It is also a multifunctional protein that plays important role in many cellular functions, viz., ribosome maturation [3], initiation of sporulation, DNA replication, chromosome partitioning [[4], [5], [6]], stress response [[7], [8], [9]], etc. Mutations on CgtA cause defects in 16S rRNA processing, ribosomal protein modifications and alter ribosomal protein levels in cell, entailing it as a major player in ribosome biogenesis either as an rRNA/ribosomal protein folding chaperone or scaffolding protein [10]. It has been suggested that ObgE, i.e., the CgtA of Escherichia coli, is involved in final checkpoint of the 50S subunit assembly under normal growth conditions. ObgE is considered to be a specialized translation factor during stress and acts as anti-association factor in presence of ppGpp and GTP [11].