The N-terminal domain of apolipoprotein B-100: structural characterization by homology modeling
© Al-Ali and Khachfe; licensee BioMed Central Ltd. 2007
Received: 09 October 2006
Accepted: 22 July 2007
Published: 22 July 2007
Apolipoprotein B-100 (apo B-100) stands as one of the largest proteins in humans. Its large size of 4536 amino acids hampers the production of X-ray diffraction quality crystals and hinders in-solution NMR analysis, and thus necessitates a domain-based approach for the structural characterization of the multi-domain full-length apo B.
The structure of apo B-17 (the N-terminal 17% of apolipoprotein B-100) was predicted by homology modeling based on the structure of the N-terminal domain of lipovitellin (LV), a protein that shares not only sequence similarity with B17, but also a functional aspect of lipid binding and transport. The model structure was first induced to accommodate the six disulfide bonds found in that region, and then optimized using simulated annealing.
The content of secondary structural elements in this model structure correlates well with the reported data from other biophysical probes. The overall topology of the model conforms with the structural outline corresponding to the apo B-17 domain as seen in the EM representation of the complete LDL structure.
Atherosclerosis is a complex disease that has been linked to many risk factors, including hyperlipidemia, dyslipidemia, high blood pressure, and endothelial dysfunction . Oxidative modification to the small low-density lipoprotein (LDL) has been dubbed the central event that initiates and propagates coronary artery diseases [2, 3], and therefore, LDL is considered a major risk factor for atherosclerosis . It was also shown that systemic inflammatory mechanisms may underlie the pathogenesis of atherosclerosis [5–7]. However, the specific structural interactions implicated in these mechanisms have not yet been elucidated.
Apolipoprotein B-100 (apo B) is the sole protein component of LDL ; however, its large size (4536 a.a.) and the limitation of current experimental techniques require that the structures of its multiple domains be analyzed separately [9, 10]. Biochemical , calorimetric , computational [12–15], and spectroscopic  approaches were used to probe the domain arrangement and characterization of the protein, but no molecular structure has ever been assigned to any of the different domains. These techniques, however, helped in the understanding of the overall arrangement of apo B on the LDL particle and the interactions that the various secondary structures have with both the lipid and aqueous phases, and in the ability to genetically engineer protein truncations that correspond to these various domains [17–20].
In this report, we describe a model structure for apo B-17 that was modeled by homology, taking the crystal structure of lipovitellin (LV) [21–23] as a template. LV – coded 1LSH in the Protein Data Bank (PDB) repository – shares more than 30% sequence similarity with the first 782 a.a. of apo B (the N-terminal 17% of the full-length sequence), a region that is rich in disulfide bonds [24, 25], essential for the secretion of the protein from hepatic cells , and behaves like an independent globular protein [19, 20]. It seemed logical to try to characterize the structure of B17 using homology modeling as a starting step towards the study of the whole structure of apo B-100.
Results and discussion
LDL has been termed as the agent provocateur of atherosclerosis. Since ApoB-100 is the sole protein component of LDL, it is expected that it plays an important role in the atherogeneity of the lipid particle. The huge size of the polypeptide hinders standard structural characterization approaches, and necessitates that it be studied in pieces, possibly correlating with the domain organization previously characterized by biochemical studies.
One disulfide pair came within binding distance after a series of minimization runs, and two pairs approached binding distance through directed (constrained) minimization, adding up to 6 disulfide bridges. However, those that were subject to constrained minimization were located within flexible loops at the surface of the protein, and thus did not cause the overall fold to change. Minimization was done in a step-wise fashion in order to explore bonding space between the sulfur groups without distorting secondary formations. Finally, a molecular dynamics simulation at 25–27 degrees Celsius was performed on the B17 molecule to allow its side chains to explore allowed conformational space.
Solvent accessibility for the buried salt bridge.
Solvent Accessibility Surface Area (Å2)
The structure of LV has been reported to contain a completely buried salt bridge formed between R547 and E574 , which ties together the two "helical sheets" in the α-domain, thereby increasing the stability of the local fold. A careful inspection of the B17 model structure revealed that a very similar salt bridge is formed between K530 and E557, which align – sequentially – with the above-mentioned residues in LV. Moreover, the solvent accessibility analysis illustrates that the involved side chains are well shielded form the aqueous medium and can therefore account for an extra stability in the α-domain of B17 that has been previously reported [19, 20].
This model provides further insight into the structural basis for the functional attributes of B-17, and constitutes a step towards the full elucidation of the multi-domain structure of full-length Apo B-100. While the current structure ensures the globular topology of the domain and its poor lipidation state, as it does not show lipid binding pockets, the biological implications of this protein – independent of its role in apo B-100 – remain to be tested in vitro and, later, in vivo, since B17 is not a naturally occurring plasma apolipoprotein. Knowing the importance of this domain in the secretion and assembly of the full-length apo B-100, we anticipate that the current structure and subsequent physiological experiments will assist in the development of novel drugs for the treatment of and protection against diseases correlated with elevated blood LDL.
Multiple sequence alignments were done using BLAST  and the alignment module of the Discovery Studio suite (Accelrys Inc., Discovery Studio 1.5, San Diego: Accelrys Inc., 2004)
The structure of B17 (residues 1–704) was modeled using MODELLER  of HOMOLOGY in insight II (Accelrys Inc., Insight Modeling Environment, Release 2000.1, San Diego: Accelrys Inc., 2002), based on the crystal structure of lipovitellin (LV), an egg yolk protein that shares over 30% sequence homology (in over 700 amino acid overlap) with B17. The secondary structure of the unstructured region was predicted using the Chou-Fasman Algorithm , the PROF methods [27, 28] and the Deep View modality . The calculation was performed using the Accelrys SeqWeb server of the GCG Wisconsin Package.
EC's were performed using DISCOVER (Accelrys Inc., CDiscover Molecular Simulator, Release 2000.1, San Diego: Accelrys Inc., 2002) and CHARMm (Version c28b)  modules in Insight II. Energy minimizations were performed using the Steepest Descent method followed by Conjugate Gradients.
MD Simulations were carried out with periodic boundary conditions using a cubic box (of appropriate size), in the Insight II package. Solvent water molecules were represented by the three-site TIP3P water model , in the NVT ensemble.
Calculations were performed using the DISCOVER force-fields CVFF and CFF91. The CHARMm force-field used in the solvation simulation was CHARMm27.
Solvent Accessible Surface Area (SASA) was calculated for individual atoms using the Structural Biology at NIH server (Structools), with a probe radius of 1.4 Å .
Solvation energy and hydrophobic interactions were calculated using the Delphi module in Insight II (Accelrys Inc., Delphi Module, Release 2000.1, San Diego: Accelrys Inc., 2002), using the CFF91 force-field. Potential maps were constructed using a grid. The dielectric value was assigned as 4 for the protein and 80 for the solvent.
The authors wish to thank Professor David Atkinson for his insightful comments and Professor Sawsan Khouri for her helpful remarks. This work has been supported in part by the American University of Beirut's Medical Practice Plan (AUB-MPP) and University Research Board (AUB-URB) funds.
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