Effects of point mutations in the binding pocket of the mouse major urinary protein MUP20 on ligand affinity and specificity

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K–adenosine triphosphatase (ATPase) α subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane’s electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis α1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain–sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 µM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump–induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

Download Full-text

Major Urinary Protein Complex of Normal Mice: Origin

Science ◽

10.1126/science.149.3687.981 ◽

1965 ◽

Vol 149 (3687) ◽

pp. 981-982 ◽

Cited By ~ 102

Author(s):

J. S. Finlayson ◽

R. Asofsky ◽

M. Potter ◽

C. C. Runner

Keyword(s):

Protein Complex ◽

Urinary Protein ◽

Major Urinary Protein

Download Full-text

Interrogating the cytoglobin binding pocket and thiol redox status with point mutations and chemical probes

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2018.10.086 ◽

2018 ◽

Vol 128 ◽

pp. S50

Author(s):

Anthony DeMartino ◽

Jesus Tejero ◽

Mark Gladwin

Keyword(s):

Point Mutations ◽

Binding Pocket ◽

Redox Status ◽

Chemical Probes ◽

Thiol Redox

Download Full-text

Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/gkt1281 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3246-3260 ◽

Cited By ~ 16

Author(s):

Sunny Sharma ◽

Jun Yang ◽

Simon Düttmann ◽

Peter Watzinger ◽

Peter Kötter ◽

...

Keyword(s):

Large Subunit ◽

Point Mutations ◽

Binding Pocket ◽

Phenotypic Characterization ◽

Deletion Mutants ◽

Chemical Modifications ◽

Gene Complementation ◽

Cellular Physiology ◽

Complex Processes ◽

Rp Hplc

Abstract RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m3U2634 and m3U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold–like methyltransferases and the point mutations in the S-adenosyl-l-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m5U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.

Download Full-text