Photon Factory Activity Report 2016 #34 (2017)

AR-NW12A/2016G678

Structure of yeast N-terminal amidase in the N-end rule pathway
Min Kyung Kim, Sun Joo Oh, Byung-Gil Lee and Hyun Kyu Song*Division of Life Sciences, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, South Korea

1Introduction
The N-end rule pathway is a universally presentubiquitin (Ub)-dependent proteolytic system that mediates
and regulates the degradation of intracellular proteinsthrough the recognition of their (destabilizing) N-terminal
residues. [1] It is conserved among both prokaryotes andeukaryotes. In this hierarchically organized pathway, Nterminal amidase converts N-terminal asparagine (Asn)and N-terminal glutamine (Gln), the tertiary destabilizing
residues of N-end rule substrates, to the secondary
destabilizing N-terminal residues aspartic acid andglutamic acid, respectively. The yeast (Saccharomycescerevisiae) N-terminal amidase Nta1 was identified as acomponent of the N-end rule pathway more than 20 years
ago [2] but its dual specificity for N-terminal Asn and Gln
residues remains uncharacterized, owing the absence ofstructural and biochemical data. The present structures ofyeast Nta1 with various N-degron peptides, andbiochemical analyses with Nta1 mutants, illuminate
specific mechanisms of the first step in the N-end rulepathway.
ExperimentyNta1 and mutants were expressed with modified pET
vector containing a N-terminal 6×His-bRIP (ribosome inactivation protein from barley) vector. [3] The overexpressed proteins were purified by Ni-NTA affinity
chromatography followed by anion exchange andadditional Ni-NTA column and size exclusion
chromatography. yNta1 was crystallized using the hanging
drop method by vapor-diffusion at 20 ºC. To cocrystallized
purified yNta1 mutants was incubated with ~ 25 fold molar
excesses of each peptide for 24 h. yNta1 and substrate
peptide complex crystal diffracted up to 2.0 ~3.0 Å. The
space group and unit cell parameters were I4122 with
a=b=134.1 Å and c=119.5 Å. Diffraction data werecollected using an ADSC quantum CCD detector at theNW12 beamline of Photon Factory. The diffraction data
were processed and scaled using the HKL2000 software
package. Phases were obtained by Selenomethionin
derivative crystal using Autosol according to the PHENIX
program suite. The initial model was manually built andrefined using COOT and PHENIX.

yNta1 has an α-β-β-α sandwich fold, which is commonly
found in members of the nitrilase superfamily. [4] yNta1
exist a monomer containing 14 β-strands, 11 α-helices, andthree 310-helices. The core region of the enzyme shows
antiparallel and parallel mixed β-sheets surrounded byhelices, and these six stranded β-sheets face each other (Fig.
1A). yNta1 makes cleft to interact with different N-degron
peptides. The cleft of the active site could be tightly fitted
to N-degrons and no complex structures showedsignificant conformational changes, in comparison withthe structures of free yNta1 (Fig. 1B).

A2

B

Fig. 1: (A) Overall structures of yeast Nta1. (B) Close-up viewof N-degron binding site and electron-density map for the boundN-degrons Asn–Val and Gln–Val dipeptide

References[1] A. Varchavsky, Cell 69, 725 (1992)[2] R.T. Baker et al., JBC 270, 12065 (1995)[3] B.-G. Lee et al. Acta Cryst. D 68, 1488 (2012)[4] M.K. Kim et al., Proc Natl Acad Sci USA 113, 12438(2016)*hksong@korea.ac.kr
3Results