Z. Hu, et al.
Journal of Structural Biology xxx (xxxx) xxx–xxx
et al., 2017; He and Bjorkman, 2011; Hu et al., 2018). It has been
shown that MR may have complicated conformational changes as pH
decreases (Hu et al., 2018), and the pH-dependent conformational
change might correlate with the ligand binding and release during re-
cycling (Gazi and Martinez-Pomares, 2009). For example, both the
CysR and CTLD4 domain of MR have shown pH-dependent ligand-
binding properties, and the collagen recognition of MR is also affected
by pH (Hu et al., 2018; Liu et al., 2001; Mullin et al., 1994). Un-
fortunately, although the pH-dependent properties of the MR family
members have been studied before by different biophysical methods
such as electron microscopy (EM), dynamic light scattering (DLS),
small-angle X-ray scattering (SAXS) (Cao et al., 2015; Dong et al., 2017;
He and Bjorkman, 2011; Hu et al., 2018), the mechanisms remain un-
clear, largely due to the lack of high-resolution structural information at
different pH.
processed using the HKL-3000 package (Minor et al., 2006). The
structures of CysR~CTLD2 were solved by molecular replacement using
the program PHASER (McCoy et al., 2007). The CysR domain, the FnII
domain and CTLD1-2 of MR (PDB code: 5XTS) were used as search
models. To determine the structure of the CysR~CTLD3 fragment (pH
5.6), CTLD1-2 of MR (PDB code: 5XTS) were used as a search model and
a partial solution was obtained by molecular replacement. Then the
heavy-atom (Se) sites were identified using program Autosol
(Terwilliger et al., 2009), and program Autobuild was used for model
building (Terwilliger et al., 2008). Coot (Emsley and Cowtan, 2004)
and Phenix (Adams et al., 2010) were used for structural refinement.
Figures were generated using UCSF Chimera (Pettersen et al., 2004).
2.3. Small-angle X-ray scattering
Here we investigated the structural basis of the pH-dependent
conformational change of human MR by solving a series of crystal
structures of MR N-terminal fragments at different pH, and also identify
the residues involved in the pH-dependent conformational change by
mutagenesis studies, revealing the molecular mechanisms of the N-
terminal fragment of MR.
The purified protein (CysR~CTLD3) with a series of concentrations
(1~10 mg/mL) at pH 6.0 was used for SAXS experiments. SAXS data
were collected at 4 °C with 20 × 1 s exposures with a PILATUS1M de-
tector at BL19U2 beamline of National Facility for Protein Science
Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility (SSRF).
Scattering data were analyzed using software package BioXTAS RAW
(
Nielsen et al., 2009) and the ATSAS program suite (Petoukhov et al.,
2
. Materials and methods
2012). Background scattering was subtracted using PRIMUS in the
ATSAS. Pair distance distribution functions of the particles P(r), radii of
gyration (Rg) and the maximum diameters (Dmax) were calculate using
GNOM in the ATSAS. Ab initio models were calculated using DAMMIF/
DAMMIN in the ATSAS with 20 runs for each experimental group,
DAMAVER in the ATSAS was used to align the ab initio models and
generate an averaging model. UCSF Chimera was used to dock the
crystal structure into the low-resolution contour of the ab initio model
(Pettersen et al., 2004). CRYSOL in the ATSAS was used to evaluate the
fitting of the back-calculated profile of crystal structure against the
experimental scattering curve.
2.1. Protein expression and purification
Constructs encoding CysR~CTLD3 (residues 1~629 of MR protein)
and CysR~CTLD2 (residues 1~490 of MR protein) with a C-terminal
xHis-tag were sub-cloned into the pFastBac vectors (Invitrogen). The
6
Sf9 cells were used for generating recombinant baculoviruses and High-
Five (Hi5) cells were used for protein production. The infected cells
were cultured in ESF921 medium (Expression Systems) for 3 days in a
27 °C humidified incubator.
For the selenomethionine protein expression, Hi5 cells were in-
fected with the recombinant baculoviruses. After 16 h, the normal
medium was replaced by the methionine-free medium (Expression
Systems). Then the selenomethionine (Sigma) was added into the media
with 100 mg/l concentration after 4~5 h, and the cells were cultured
for 48 h before supernatant collection.
The supernatants were buffer-exchanged with 25 mM Tris, 150 mM
NaCl at pH 8.0 by dialysis, then applied to Ni-NTA chromatography (Ni-
NTA Superflow, Qiagen). The eluted proteins were further purified by
gel filtration chromatography with a HiLoad Superdex 200 16/600 pg
column (GE Healthcare).
2.4. Size-exclusion chromatography
Before the SEC experiments, the purified proteins were buffer-ex-
changed with the running buffers (150 mM NaCl, 50 mM PBS at pH 7.4
or pH 6.0). For the high ionic strength assays, buffer (500 mM NaCl,
50 mM PBS at pH 7.4 or pH 6.0) and buffer (1 M NaCl, 50 mM PBS at pH
7.4 or pH 6.0) were used. SEC was performed on a HiLoad Superdex
200 16/600 pg column (GE Healthcare) with a flow rate of 1.0 ml/min.
3. Results
2.2. Crystallization and structural determination
3.1. The N-terminal fragment CysR~CTLD2 of MR adopts an L-shaped
conformation in the crystals at different pH
The purified proteins (CysR~CTLD3 and CysR~CTLD2) were
buffer-exchanged into 5 mM Tris, 100 mM NaCl (pH 7.4) at 10 mg/mL
concentration (measured by UV absorption at 280 nm). Crystal
screening was performed at 18 °C by hanging-drop vapor diffusion
method using 48-well plates (Hampton Research, Molecular
Dimensions, Wizard). The crystals of the CysR~CTLD3 fragment were
grown in a solution containing 0.5 M ammonium sulfate, 0.1 M sodium
citrate tribasic dihydrate (pH 5.6), 1.0 M lithium sulfate monohydrate
It has been shown that the N-terminal fragment CysR~CTLD3 of MR
undergoes a pH-dependent conformational change as pH decreases (Hu
et al., 2018). In order to explore the mechanism behind the con-
formational change, we screened the crystallization conditions for the
CysR~CTLD2 and the CysR~CTLD3 fragments at pH ranging from pH
4.0 to pH 8.5 (Fig. 1A), and were able to obtain diffractable crystals of
CysR~CTLD2 at pH 4.0, pH 4.6, pH 6.0 and pH 8.5 (Fig. 1B and
Table 1) as well as crystals of CysR~CTLD3 at pH 7.0 and pH 5.6
(Fig. 3A). Among them, the crystal structure of CysR~CTLD2 at pH 6.0
and the crystal structure of CysR~CTLD3 at pH 7.0 have been solved
previously (Fig. S1) (PDB code: 5XTW and 5XTS, respectively) (Hu
et al., 2018). In all these crystal structures, CysR~CTLD2 adopts an L-
shaped conformation, and the structural superposition shows that the L-
shaped conformation is well conserved at both acidic and basic condi-
tions (Fig. 1C) (Hu et al., 2018). These results suggest that the first four
N-terminal domains of MR may have a relatively rigid conformation,
similar to Endo180, another member in the MR family (Paracuellos
et al., 2015).
(
final pH 5.3~5.6). The crystals of the CysR~CTLD2 fragment at dif-
ferent pH were grown in the following conditions, respectively: 1)
.2 M potassium sodium tartrate tetrahydrate, 0.1 M Tris (pH 8.5) (final
1
pH 8.5~9.0); 2) 0.01 M cobalt chloride hexahydrate, 1.0 M 1,6-
a
Hexanediol, 0.1 M sodium acetate trihydrate (pH 4.6 ) (final pH
4
.6~4.8); 3) 2.0 M sodium chloride, 0.1 M sodium acetate trihydrate
b
(
pH 4.6 ) (final pH 4.4~4.6); 4) 1.0 M lithium chloride, 10% (w/v) PEG
6000, 0.1 M citric acid (pH 4.0) (final pH 4.0).
Diffraction data were collected using a PILATUS6M detector at
BL18U beamline of National Facility for Protein Science Shanghai
NFPS) at Shanghai Synchrotron Radiation Facility (SSRF) and
(
2