Conceived and designed the experiments: AGT CP MIS LL. Performed the experiments: AGT CP MS MN RDB TK LL. Analyzed the data: WHL HS LL. Wrote the paper: HS LL.
The authors have received funding from the following commercial sources: GlaxoSmithkline, Merck & Co., Inc. and Novartis. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.
We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.
Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.
The START domain is a ubiquitous conserved module for binding and transporting lipids
Structural analyses of START domains from groups 1–3 have provided detailed insights into how these proteins sequester specific lipids
Group | Protein | Ligand | PDB entry |
|
STARD1 | cholesterol |
3P0L; ligand-free (this study) |
STARD3/MLN64 | cholesterol |
1EM2; ligand-free |
|
|
STARD4 | cholesterol |
1JSS (mouse); ligand-free |
STARD5 | cholesterol, 25-hydroxycholesterol |
2R55; ligand-free (this study) | |
STARD6 | cholesterol |
- | |
|
STARD2/PCTP | phosphatidyl choline |
1LN1; DLP complex |
STARD7 | phosphatidyl choline |
- | |
STARD10 | phosphatidylcholine/ethanolamine |
- | |
STARD11/CERT | ceramides |
2E3R; C18-ceramide complex |
|
|
STARD8 | charged lipid? | - |
STARD12 | charged lipid? | - | |
STARD13 | charged lipid? | 2PSO; ligand-free (this study) | |
|
STARD14 | fatty acid? | 3FO5; PEG complex |
STARD15 | fatty acid? | - | |
|
STARD9 | ? | - |
DLP, 1,2-dilinoleoyl-SN-glycero-3-phosphocholine.
DAG, diacylglycerol.
H15, 3-hydroxy-1-(hydroxymethyl)-3- phenylpropyl]pentadecanamide.
PEG, pentaethylene glycol.
We used a structural genomics approach to human START domain containing proteins. Based on previously published crystal structures multiple expression constructs were designed for STARD1, STARD5, STARD7–11, STARD13 and STARD14. Following recombinant protein production in
Condition | STARD1native | STARD5native | STARD13native | STARD14native | STARD14SeMet |
|
200 mM Ca-acetate | - | 125 mM NaCl | 200 mM MgCl2 | 200 mM NaSCN |
|
40% PEG-300 | 10% PEG-6000 | 20% MPD | 25% PEG-3350 | 20% PEG-3350 |
|
100 mM Na-cacodylate | 100 mM HEPES | 100 mM Tris-HCl | 100 mM Bis-Tris | |
|
6.5 | 7.0 | 8.0 | 5.5 | 6.9 |
|
Cholesterol | - | - | - | - |
|
4°C | 4°C | 4°C | 4°C | 4°C |
|
Sitting drop | Sitting drop | Hanging drop | Sitting drop | Sitting drop |
|
0.2/0.2 | 0.8/0.4 | 0.8/0.2 | 0.2/0.2 | 0.2/0.4 |
|
50% PEG-300 | 20% BD | 40% MPD | 20% Glycerol | 18% Glycerol |
Protein | STARD1 | STARD5 | STARD13 | STARD14 | STARD14 SeMet pk | STARD14 SeMet ip | STARD14 SeMet rm |
|
ESRF ID29 | ESRF ID14.4 | ESRF ID14.4 | ESRF ID14-2 | BESSY BL14-2 | BESSY BL14-2 | BESSY BL14-2 |
|
0.97948 | 1.03992 | 1.03992 | 0.93300 | 0.97973 | 0.97985 | 0.97201 |
|
P63 | P65 | P43 | C2221 | P31 | P31 | P31 |
|
|||||||
|
144.130, 144.130, 101.030 | 62.87, 62.87, 214.93 | 78.24, 78.24, 212.72 | 52.44, 130.08, 165.23 | 69.41, 69.41, 97.25 | 69.41, 69.41, 97.25 | 69.41, 69.41, 97.25 |
|
90, 90, 120 | 90, 90, 120 | 90, 90, 90 | 90, 90, 90 | 90, 90, 120 | 90, 90, 120 | 90, 90, 120 |
|
15-3.4 (3.5-3.4) | 20-2.5 (2.6-2.5) | 20-2.8 (2.9-2.8) | 20-2.0 (2.1-2.0) | 50-2.3 (2.36-2.3) | 50-2.3 (2.36-2.3) | 50-2.3 (2.36-2.3) |
|
0.173 (0.797) | 0.102 (0.671) | 0.038 (0.556) | 0.055 (0.545) | 0.040 (0.468) | 0.037 (0.471) | 0.036 (0.533) |
|
19.8 (4.8) | 23.3 (5.9) | 15.2 (3.7) | 15.3 (2.6) | 10.1 (1.6) | 10.8 (1.6) | 11.5 (1.4) |
|
98.6 (100) | 99.4 (99.5) | 99.5 | 98.1 (99.5) | 98.5 (97.9) | 98.4 (97.8) | 98.4 (97.5) |
|
22.6 (22.8) | 22.3 (22.1) | 7.5 (7.7) | 3.9 (3.9) | 1.6 (1.6) | 1.6 (1.6) | 1.6 (1.6) |
*Values for the highest resolution shell are shown in parentheses.
†Rmerge = Σi | Ii−〈I〉 |/Σ 〈I〉, where I is an individual intensity measurement and 〈I〉 is the average intensity for this reflection with summation over all data.
Despite the low sequence identity among the START domains (
Protein sequences were aligned based on the available crystal structures as detailed in
(A) Cartoon of the START domains studied here in context of the respective full-length proteins (drawn approximately to scale). (B–E) Side-by-side comparison of human STARD1, -5, -13, and -14 in a similar orientation. All START domain structures are colored from the N-terminus (blue) to the C-terminus (red) and the linker to the N-terminal thioesterase domain of STARD14 (panel E) is shown in grey. (F) Stereo view of a superposition of the backbone traces of the four crystal structures shown in panels B through E (blue, STARD1; red, STARD5; cyan, STARD13; yellow, STARD14). The view is that of panels B–E with an approximately 90° rotation downward toward the viewer.
We solved the crystal structure of STARD1, a member of the StAR group, at a relatively low resolution of 3.4 Å (
(A) Packing of STARD1 in the crystal lattice with the tube formed around the 63-axis. Monomers A–D in the asymmetric unit are colored individually, and symmetry generated molecules around the axis are shown. (B) STARD13 structure displaying the N-terminal helix swap with the adjacent protein molecule in the crystal. Two monomers (blue and white) are shown and the N-terminal helix of a third monomer is shown in magenta. Side chains are displayed for one of the C-terminal helices.
Homology modeling and subsequent ligand docking trials were previously studied in an effort to understand biological functions of STARD1
Cholesterol was included in the crystallization buffer. However, additional density which was observed in the cavity did not match the expected density of cholesterol. We believe that the cavity was either empty or partially occupied by a small ligand derived from the expression host or from the crystallization solution.
STARD5
The structure of human STARD13, a member of the RhoGAP group, is also most similar to mouse STARD4, with an rmsd of 1.8 Å for 164 Cα-atoms. The largest difference between STARD13 and other START domain structures lies in the N-terminal helix, which in STARD13 is swapped with the adjacent protein in the crystal (
The asymmetric unit of the crystal of STARD14/ACOT11 contains a dimer. The large buried surface area between the monomers (900 Å2 per monomer, as determined by the PISA server
The unique N-terminal helix (α0) of STARD14 (
Human START domains share a significant but low sequence identity (as low as 14%). As a consequence, homology-based sequence alignment methods make prediction of the positions of critical residues within the physiological START domain structures challenging. We generated a structure based sequence alignment by superposing all known START domain structures, and using this 3D alignment as a basis for aligning the sequences of the human START domain classes. This method yielded an improved alignment, and displayed similarities between individual proteins that have been overlooked by homology based methods (
Notably, there are three absolutely conserved residues (Trp96, Trp147 and Arg217; STARD1 numbering) and a highly conserved Asp183 that is replaced by the similar glutamate only in STARD4 (
(A) Positions of strictly conserved residues, as identified by the alignment shown in
Lipoid CAH is linked also to other mutations in the STARD1 encoding gene. Some of these mutations lead to premature stop codons, while others change the protein activities and lipid binding capabilities
Cavity sizes in the known START proteins vary from 873 Å3 to 2297 Å3 (based on the molecular surfaces of ligand bound as well as ligand free structures). STARD14 has clearly smallest cavity of the family. Cholesterol binding START domains have cavity sizes of 1014–1122 Å3, which is close to the size of the natural ligand. The largest cavity is observed for STARD2, which also binds larger ligand than other characterized members of the family (
Side-by-side comparison of the structures that have been solved with a ligand bound in the cavity: (A) STARD2 (1LN1) with 1,2-dilinoleoyl-SN-glycero-3-phosphocholine (DLP); (B) STARD11 (2E3R) C16-ceramide; (C) STARD14 (3FO5) with PEG or putative fatty acid.
Inspection of the ligand cavity of ligand free STARD1 suggests Glu169, Arg188, Leu199 and His220 as key residues in cholesterol binding. These side chains will likely change conformation upon ligand binding. Notably only His220 is conserved among the cholesterol binding members. Ligand docking predicted cholesterol binding to STARD1 involves a hydrogen bond between the cholesterol hydroxyl and either the Arg188 side chain or the backbone carbonyl of Leu199
In order to understand ligand binding in STARD5, we docked a cholesterol molecule to the binding cavity of the STARD5 structure. All the top ranked binding modes had cholesterol in the so-called “IN” conformation, with the hydroxyl group of cholesterol pointing towards the cavity (
(A) Model of cholesterol binding to STARD5. (B) Lipid binding cavity of STARD13, with the cavity inner surface indicated in the background (magenta). Side chains that are conserved and structurally complementary among STARD13 (blue) and STARD2 (grey) are shown as sticks. The C-terminal helix of STARD13 is shown as a blue cartoon to illustrate clashes with the STARD2 ligand. (C) Ligand binding to STARD14. Difference density is contoured at 2σ around the modeled PEG molecule in monomer B to show the elongated shape with density for the head group resembling a carboxyl group.
STARD5, in contrast to STARD1, can also bind 25-hydroxycholesterol
The natural ligand of STARD13 is unknown. We looked to identify possible ligands based on the STARD13 side chains at the positions that correspond to those involved in lipid binding in other family members. From the crystal complexes of STARD11 and ceramides we know that Arg442144 and Glu446148 are the only conserved residues between the proteins making contacts with ceramide, Glu446148 being the most critical
Notably, the ligand binding cavity of STARD13 is smaller than that of STARD2 and elongated, with a small maximum diameter (
The lipid binding cavity of STARD14 is rather hydrophobic as it is lined by phenylalanine, valine, leucine and isoleucine side chains. The cavity also contains patches of charged and hydrophilic residues, possibly making specific interactions with an unknown ligand. Inside the STARD14 cavity we observed a continuous electron density that by its shape resembles a fatty acid (
The STARD14 structure is expanded in comparison to the empty START domain structures, although the C-terminal helices are in a similar position as the C-terminal helix of STARD2 solved in complex with phosphatidylcholine. Possibly BFIT1 and BFIT2, the isoforms of STARD14, could have different ligand specificity. The crystallized form (BFIT2) contains two helices at the C-terminus whereas BFIT1 probably has only one, as seen in other START domains (
A structure based alignment reveals important features within the START domain subfamilies, and highlights critical conserved residues involved in ligand binding. Of particular interest in the scope of this paper are STARD13 and STARD14, the family members for which the ligands are not known. STARD13 Arg974/STARD14 Arg449144 is highly conserved (
The crystal structures reported here help to gain a family wide understanding of the structural determinants within the START domain family. Use of these results to create a structure-based alignment helped to determine the conserved features within the family which are overlooked by sequence homology based methods. Many human START domains have unknown functions and their apo-structures form a structural basis for ligand identification thereby providing new leads to biological functions. All structures reported are relevant to disease. They are down- (STARD13) or up-regulated (STARD5) in cancers, mutations in them result in metabolic disorders (STARD1) or they are linked to obesity (STARD14;
The cDNAs coding for full-length human STARD1, STARD5, and STARD14 were obtained from the Mammalian Gene Collection (accession codes BC010550, BC004365 and BC093846, respectively). The cDNA encoding full-length human STARD13 was PCR amplified from pooled human brain, liver, placenta, and thymus cDNA libraries (Ambion). The sequence coding for residues STARD1T66-R284, STARD5A6-E213, STARD13E51-I264, and STARD14R339-L594 were subcloned into expression vector pNIC-Bsa4 by ligation-independent cloning. The resulting expression constructs contained a hexahistidine tag and a TEV-protease cleavage site (MHHHHHHSSGVDLGTENLYFQS) at the N-terminus.
Each expression construct was transformed into
Crystallization was done by the sitting or hanging drop vapor diffusion method. Proteins in gel filtration buffer were mixed with reservoir solution (see
Synchrotron radiation datasets were collected at ESRF, Grenoble, France and at BESSY, Berlin, Germany. Data sets were indexed, scaled, and reduced using XDS (
Details of the structures are given in
Protein | STARD1 | STARD5 | STARD13 | STARD14 |
|
3P0L | 2R55 | 2PSO | 3FO5 |
|
1EM2 | 1JSS | 1JSS | - |
|
- | - | - | PEG |
|
0.257/0.287 | 0.232/0.276 | 0.210/0.244 | 0.202/0.251 |
|
4 | 2 | 3 | 2 |
|
||||
|
6373 | 3292 | 4477 | 3813 |
|
- | - | - | 56 |
|
- | 26 | - | 168 |
|
||||
|
128 | 78.8 | 90.1 | 39.6 |
|
- | - | - | 40.08 |
|
- | 51.1 | - | 28.5 |
|
||||
|
0.002 | 0.012 | 0.011 | 0.013 |
|
0.512 | 1.375 | 1.454 | 1.386 |
|
||||
|
92.3 | 93.6 | 90.1 | 98.7 |
|
7.7 | 6.4 | 9.0 | 1.3 |
|
- | - | 0.9 | - |
†Rwork is defined as Σ ||Fobs |−|Fcalc || Σ | Fobs |, where Fobs and Fcalc are observed and calculated structure-factor amplitudes, respectively.
‡Rfree is the R factor for the test set (5–10% of the data).
The STARD5 structure was solved by molecular replacement with MOLREP
STARD13 was solved by molecular replacement with MRBUMP
The STARD14 structure was solved using Solve
Models were validated with Molprobity
Docking of cholesterol to STARD5 structure was done with ICM (Molsoft). Residues surrounding the cavity were selected to indicate the binding site and initial docking of cholesterol was done keeping the residues fixed. Best conformations were energy minimized with ICM and the residues around the docked ligand were optimized.
(ICB)
(PDF)
We gratefully acknowledge the beam line scientists at the BESSY and ESRF synchrotron radiation facilities for expert assistance.