of the wtTAP structure (2iuc9) without any ligands.
The manual refinement was performed in Coot.30
The main chain was fitted correctly from the wtTAP
template, and side chains needed only small correc-
tions. H135 residues were mutated to the correct
residues from the initial maps, and the loop region
(residues 315–320) was built manually starting with
the highest resolution H135D-TAP structure. It was
clear that the active site contained three metal ions.
The identity of each was determined by comparison
of the refinement rounds in Phenix31 and inspection
of the standard and anomalous (H135D-Zn2þ) differ-
ence electron density maps. The final round of
refinement included eight TLS groups identified af-
ter model analysis at the Translation/Libration/
Screw Motion Determination server32,33 (Supporting
Information Fig. 1). The groups were defined equal
in both monomers. The refinement statistics are pre-
sented in Table I. The final structures are made
publicly available at the protein data bank with sub-
mission codes 2w5w (H135D-3Zn2þ-TAP), 2w5v
(H135D-2Zn2þMg2þ-TAP), and 2w5x (H135E-TAP)
for the structures and r2w5wsf, r2w5vsf and
r2w5xsf for the corresponding structure factors.
were collected every 5 min in 20 mM Tris-Cl pH 7.6,
10 mM MgCl2, 200 mM NaCl, and treatment
stopped by incubation on ice for 30 min. The remain-
ing activity in the samples was then measured at
25ꢀC. Reported values are the average of at least
three measurements. The standard deviations do not
exceed 5%.
Differential scanning calorimetry
Measurements were performed using a MicroCal
MCS-DSC instrument at a scan rate of 90 K/hr and
under 2 atm pressure of nitrogen. Before the analy-
sis, samples were dialyzed overnight against 30 mM
3-(N-morpholino) propanesulfonic acid pH 7.5, which
was also used for baseline. Protein concentration for
the DSC measurements was determined by the
bicinchoninic acid protein assay (Pierce), and was
set to ꢃ4 mg/ml. Calorimetric enthalpies (DHcal
)
were calculated from the area of the transitions, lim-
ited by a linear baseline drawn between the start
and the end of the transitions and normalized for
protein concentration. Datapoints more than 70ꢀC
were excluded due to the exothermic signal drift
caused by aggregation.
Enzymatic assay
Specific activity was determined at 25ꢀC in 1M di-
ethanolamine-Cl pH 10, 10% glycerol, 10 mM MgCl2,
and 1 mM ZnCl2, with 5 mM p-nitrophenyl phos-
phate (Sigma). The amount of released product, p-
nitrophenolate, was measured using a SpectraMax
M5 96-well plate reader (Molecular Devices) at the
alkaline pH absorption peak, 405 nm.
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Weakly associated metals were removed through
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pH 7.6 for 24 hr at 4ꢀC. After the dialysis, we added
separately or in various combinations 0.1–10 mM
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