To overcome the limitations of other expression systems such as missing glycosylation (Escherichia coli
), poor yield (natural sources) or high expense (chemical synthesis), human saposin C was overexpressed in the methylotrophic yeast P. pastoris
. The expression construct included a C-terminal His6
tag which was not cleaved off prior to crystallization. It has previously been shown that human saposin C expressed with a C-terminal His tag is fully functional, as assayed by binding to phosphatidylserine and activation of glucocerebrosidase (Qi & Grabowski, 2001
; Qi et al.
). Compared with E. coli
, overexpression in P. pastoris
offers the additional benefit of extremely facilitated downstream purification since the cells secrete mainly the overexpressed protein. Thus, saposin C could be directly captured from the medium. The protein expression and purification yield was about 40 mg of protein per litre of medium, which is comparable to the yield typically obtained with an E. coli
The expectation that overexpression in yeast cells also resulted in glycosylation of Asn22 was not fulfilled. Analysing the sample by MALDI–TOF revealed a peak at 10 252.2 Da that agreed well with the length of the cloned sequence including the His6
tag with an additional arginyl residue but without glycosylation. However, glycosylation is neither essential for binding to phospholipids nor for activation of lysosomal hydrolases (Hiraiwa et al.
; Vielhaber et al.
). Four other peaks were found at weights incrementally decreasing by 137 Da, indicating that the histidyl residues were either partially cleaved off or only partially expressed.
Three different crystal forms of human saposin C were obtained using two different precipitating agents. Hexagonal crystals were obtained using 2-propanol as a precipitant and grew as needles up to a length of 750 µm (Fig. 1
a). The Bravais lattice of the crystals is primitive hexagonal with point group P622. However, the analysis of systematic absences along 00l was ambiguous and leaves the space group open to be either P6322 or one of the enantiomeric space groups P6122 and P6522. Determination of the true space group must await structure determination.
(a) Hexagonal crystals of human saposin C with a maximum length of 750 µm. (b) Tetragonal crystals with a maximum length of 300 µm. (c) An orthorhombic crystal of 500 µm in the longest direction.
The tetragonal crystal forms grew in the presence of pentaerythritol ethoxylate 15/4. Typical crystals obtained from these conditions are shown in Fig. 1(b). The needle-like crystals reached a maximum length of 300 µm, contained two molecules in the asymmetric unit and have unit-cell parameters a = 48.9, c = 154.3 Å. From the systematic absences the space group could be determined to be either P41212 or P43212.
Whereas the tetragonal crystals were obtained in sitting drops using pentaerythritol ethoxylate 15/4 as the precipitant, an orthorhombic crystal form was obtained in a hanging-drop vapour-diffusion setup using increased pentaerythritol ethoxylate 15/4 concentrations and magnesium chloride as an additive. Typical crystals are shown in Fig. 1(c). The crystal dimensions reached approximately 500 × 300 × 200 µm. No flash-freezing protocol could be established for these crystals and thus their diffraction had to be measured at room temperature using a rotating-anode source. The unit-cell parameters obtained were a = 57.0, b = 93.5, c = 88.3 Å. The space group could be unambiguously determined as C2221 from systematic absences. Data-collection statistics for each of the three different crystal forms are compiled in Table 1.
Data-collection statistics of the three different crystal forms
Interestingly, phase determination by molecular replacement using various approaches as implemented in AMoRe
(Vagin & Teplyakov, 1997
) and Phaser
) including ensemble searches failed for all saposin C crystal forms using either the SapC NMR structures (PDB codes 1m12
) or structures of other SLPs, such as the crystal structure of granulysin (PDB code 1l9l
) or the NMR structure of NKL (PDB code 1nkl
), as search models. Since data-collection statistics gave no indication of twinning and since the identity of the saposin protein has been unambiguously established by overexpression in P. pastoris
, MALDI–TOF mass spectrometry and SDS–PAGE analysis, we conclude that the failure of structure determination by molecular replacement arises from considerable conformational differences between the search models and the crystallized saposin C. Comparison of the two known NMR structures of saposin C has already demonstrated flexibility in this protein (de Alba et al.
; Hawkins et al.
). Whether each of the three different crystal forms described here represents a distinct conformational state remains to be seen. Based on the two NMR structures, we are currently calculating theoretical intermediate structures using Cartesian interpolation (Vonrhein et al.
) and normal-mode analysis (Suhre & Sanejouand, 2004
) in order to generate improved search models.
A second strategy we are pursuing to determine the structure of saposin C is to obtain experimental phases by exploiting the anomalous signal of the eight S atoms (three disulfide bridges, two methionine residues) in the protein. For this type of SAD experiment the hexagonal crystals were used to collect highly redundant data sets at an X-ray wavelength of 1.8786 Å in order to maximize the anomalous signal of sulfur (Table 1). We expect that this and future measurements with further improved data quality will allow phase determination by sulfur SAD.
From structure determination of the three different crystal forms, we expect to obtain a detailed picture on the dynamic behaviour of human saposin C and likely of other members of the saposin-like family. Determining structures of saposin C in different conformational states by X-ray crystallography may ultimately define the trajectory of its opening motion, which is a prerequisite and specificity-determining factor for substrate interaction in the whole family of SLP proteins.