Crystal structural analysis of human serum albumin complexed with hemin and fatty acid
© Zunszain et al; licensee BioMed Central Ltd. 2003
Received: 06 May 2003
Accepted: 07 July 2003
Published: 07 July 2003
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© Zunszain et al; licensee BioMed Central Ltd. 2003
Received: 06 May 2003
Accepted: 07 July 2003
Published: 07 July 2003
Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSA-heme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex.
We present here the crystal structure of a ternary HSA-hemin-myristate complex, formed at a 1:1:4 molar ratio, that contains a single hemin group bound to subdomain IB and myristate bound at six sites. The complex displays a conformation that is intermediate between defatted HSA and HSA-fatty acid complexes; this is likely to be due to low myristate occupancy in the fatty acid binding sites that drive the conformational change. The hemin group is bound within a narrow D-shaped hydrophobic cavity which usually accommodates fatty acid; the hemin propionate groups are coordinated by a triad of basic residues at the pocket entrance. The iron atom in the centre of the hemin is coordinated by Tyr161.
The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA. The details of the hemin-binding environment of HSA provide a structural foundation for efforts to modify the protein and/or the heme molecule in order to engineer complexes that have favourable oxygen-binding properties.
In the human body heme may be released into the circulation during enucleation of erythrocytes or through hemolytic injury. Free heme is immediately oxidized to the ferric state, hemin, which is potentially toxic since it may intercalate in lipid membranes and catalyze the formation of hydroxyl radicals and the oxidation of low density lipoproteins . This toxicity is largely averted though the scavenging action of hemopexin, a protein that circulates in plasma (~17 μM), binds hemin with extremely high affinity (KD < 1 pM) and transports it to various tissues – primarily liver cells – for intracellular catabolism .
Human serum albumin (HSA), the most abundant plasma protein (~640 μM) also has a high affinity for hemin. The dissociation constant for the interaction was determined to be ~10 nM in a 3:5 (v/v) mix of dimethyl sulfoxide and water  but the interaction is likely to be even tighter in a more aqueous medium. HSA may provide a reserve binding capacity for situations when hemopexin becomes saturated. The protein is principally characterized by its remarkable ability to bind a broad range of hydrophobic small molecule ligands including fatty acids, bilirubin, thyroxine, bile acids and steroids; it serves as a solubilizer and transporter for these compounds and, in some cases, provides important buffering of the free concentration . HSA also binds a wide variety of drugs in two primary sites [5, 6] which overlap with the binding locations of endogenous ligands. The protein is a helical monomer of 66 kD containing three homologous domains (I-III) each of which is composed of A and B subdomains . Despite the internal structural symmetry, the three domains have different capacities for binding fatty acids [7–10], thyroxine  and drugs [5, 8, 12, 13]. Binding studies indicate a single site for hemin in subdomain IB  which corresponds to a binding site for fatty acids [8–10].
The ability of HSA to bind hemin has stimulated efforts to develop HSA-heme complexes that mimic the reversible oxygen-binding properties of heme proteins such as myoglobin and hemoglobin. One difficulty with this approach is that even if HSA-bound hemin is reduced to heme, stable and reversible oxygen binding by the complex cannot be observed (reviewed in [15, 16]), because the heme binding site does not provide a molecular environment sufficiently similar to that found in myoglobin or hemoglobin. However, recent work has shown that artificial heme derivatives associate with HSA and provide significant oxygen-binding capacity [15, 17–21]. Although such compounds have extensive modifications to the porphyrin ring and are not expected to bind to the protein in the same fashion as heme, they represent a promising line of inquiry for the development of artificial blood substitutes.
In order to advance our understanding of the heme-binding properties of HSA and the development of viable blood substitutes based on HSA-heme complexes, we have solved the crystal structure of a HSA-hemin-myristate complex. The structure reveals a previously undetected conformational state of the protein and provides a detailed view of the heme binding site. Our results are compared with a recently reported structure for methemalbumin .
Data collection and refinement statistics
Resolution range (Å)
R merge (%) 2
R model (%) 3
R free (%) 4
r.m.s deviation from ideal bond lengths (Å)
r.m.s deviation from ideal angles (°)
Average B-factor (Å 2 )
The electron density clearly indicates the binding configuration of the porphyrin ring and the two propionate groups of the bound hemin (Figure 1b), though it is possible that the molecule binds in two overlapping orientations that are related by a two-fold rotation (180°) about its centre. In either of the two orientations the propionate groups appear to be able to make the same interactions with the protein (to residues R114, H146 and K190) but the asymmetric location of the vinyl groups projecting from the hydrophobic end of the hemin molecule would occupy different positions. From the structure it appears that both configurations of the vinyl group could be accommodated within the binding pocket but may result in small adjustments of sidechains lining the pocket and of the exact position of the porphryin ring and its iron centre. Even at higher resolution, this ambiguity is not resolved  and it may therefore reflect a genuine degeneracy in hemin binding.
The density for the fatty acids is generally rather weak, indicating partial occupancy as would be expected given the 4:1 mole ratio of fatty acid to HSA used to prepare the complex. Although there is usually clear density to indicate the position of the fatty acid carboxylate moiety in each binding pocket, the density for the methyl end of the methylene tail is weak or absent. As a result, only the visible portion of the fatty acid tails has been included in the refined model.
It is conceivable that the difference in the orientation of domain I relative to domain II in the HSA-hemin-myristate and other HSA-fatty acid complexes is also due to changes in the crystal-packing environment between the two crystal forms (P21212 and C2). However, consideration of the structure suggests that it is more likely to be due to differences in the level of bound fatty acid (Figures 2c and 2d). The crystal structure of the HSA-myristate complex – prepared using a high myristate:HSA mole ratio – showed evidence for two fatty acid molecules bound in a tail-to-tail configuration in the contiguous fatty acid sites 2 and 2' which straddle the interface between domains I and II. Occupancy of site 2 is believed to be primarily responsible for driving the conformational change between these two domains (Figure 2d). In the HSA-hemin-myristate complex, there is no evidence for fatty acid binding to site 2' and indeed the density for the fatty acid bound to site 2 is relatively weak, probably as a result of the reduced occupancy (Figure 2c). Thus it may be that binding to site 2' is required to drive the more extensive conformational change between domains I and II. Although fatty acids longer than C14 are not observed to occupy site 2', they nevertheless appear to drive the "full" conformational change; this is most likely because the methylene tail of a long-chain fatty acid bound to site 2 begins to occupy site 2' [9, 10] and may therefore play the same structural role as the binding of a second molecule of a shorter-chain fatty acid to site 2'. An independent crystallographic study on hemin binding to HSA in the presence of myristate reportedly obtained a conformation very similar to that found for HSA-myristate at very high myristate:HSA mole ratios , but the amount of myristate used was not given and the coordinates are not yet available to allow a direct comparison with our present structure . Whatever the precise mechanism underlying the fatty-acid induced conformational changes, there is no evidence to suggest a connection between binding of hemin and the new conformational stage identified in the present work. Moreover, it is likely that the hemin-binding site itself is not affected by fatty acid induced conformational changes.
Hemin binds to a hydrophobic, D-shaped cavity in subdomain IB. The hydrophobic porphyrin ring is essentially buried in the core of the subdomain with the propionate groups located at the wide entrance to the pocket where they can interact with solvent and several basic amino acid sidechains (see below). In the absence of ligand this cavity is partially occluded by two tyrosine residues (138 and 161) which stack on top of one another . Upon hemin binding both sidechains undergo χ1 rotations of around 90° thereby opening up the binding pocket and helping to clamp the ligand in place. Very similar rotations of these sidechains were observed with fatty acid binding ; indeed there is complete overlap between the positions of fatty acid and hemin when bound in this site. Other sidechains, including Leu139, His146, Ile142 and Leu154, exhibit modest adjustments upon heme binding.
All of the structural components of subdomain IB, including the polypeptide linker connecting it to subdomain IA and helices 7–10, contribute to the hemin binding site. The hemin group binds with its plane oriented at about 30° to the directional axes of helices 8–10; this orientation is determined by the packing of the hemin group against the residues lining the interior wall of the pocket. With the exception of Tyr138 and Tyr161, these are entirely hydrophobic sidechains. The hydroxyl group of Tyr138 is relocated to the exterior of the domain where it interacts with solvent, but the hydroxyl group of Tyr161 makes a direct interaction with the central Fe3+ atom, providing a fifth point of coordination (Figure 3b). The entrance to the pocket, which is rather open, faces towards the interdomain cleft and contains three basic residues that coordinate the two propionate groups on hemin. Lys190 adopts a central position and makes salt-bridges to both propionate groups (2.7 and 3.0 Å); His146 provides a second electrostatic interaction with one heme carboxylate (3.0 Å) while the guanidinium group of Arg114 interacts with the other (2.8 Å). Arg117, which lies towards the top end of the pocket entrance and invariably interacts with fatty acids which bind at this site , is not involved in coordinating the propionate groups of the hemin. The electrostatic interactions to the propionate groups are clearly important for hemin binding; the number of residues involved in coordinating the propionate groups is fewer that the seven observed in hemopexin  but more than the one or two basic residues found to fill this role in myoglobin or hemoglobin (Figure 3c). At the opposite end of the pocket there is a small opening which is large enough to admit solvent molecules.
In terms of the general hydrophobicity of the pocket and the coordination of the propionate groups by basic residues, HSA appears to have similar features to the heme binding site on myoglobin or hemoglobin. However, although both proteins are helical there is little similarity in the architectures of their heme binding pockets. In particular, HSA lacks the pair of histidine residues that serve to enhance and regulate the relative oxygen- and carbon monoxide-binding properties of myoglobin and hemoglobin. Modification of the heme-binding pocket on HSA or of the heme-group itself is likely to be required to achieve a protein-heme complex capable of reversible oxygen binding. Although various heme derivatives have been produced that exhibit reversible oxygen binding in association with HSA [19–21, 26], these contain sizeable additions to the porphyrin ring structure and are unlikely to bind to subdomain IB on HSA in the same configuration that has been observed for hemin. Nevertheless, the detailed architecture of the heme-binding pocket as revealed by this crystallographic study will facilitate the design of new heme derivatives that will be better accommodated in this site and may therefore bind with higher affinity. It will also enable the design of mutagenesis experiments to tailor the pocket to bind modified hemes.
The structure of the HSA-hemin-myristate complex reported here reveals a new conformational state of the protein, one that is intermediate between unliganded HSA and the saturated HSA-fatty acid complex. The observation of this conformation is likely due to the use of subsaturating amounts of fatty acid in preparing the crystals and provides new insights into the mechanism of conformational change.
The single hemin-binding pocket in subdomain IB of HSA consists of a deep hydrophobic slot that provides three basic residues at its entrance to coordinate the two propionate groups of the hemin. This provides a framework for future attempts to engineer the protein and heme derivative in order to generate high-affinity HSA-heme complexes which bind oxygen reversibly and may serve as effective artificial blood substitutes.
Recombinant HSA (Recombin®), kindly provided by Delta Biotechnology (Nottingham, UK), was defatted and purified by gel filtration to be dimer-free in 50 mM potassium phosphate, 50 mM NaCl, pH 7.0 to a concentration of 110 mg/ml (1.67 mM) essentially as described previously . Immediately prior to use hemin was dissolved at 10 mM in dimethylsulfoxide (DMSO) in a foil-wrapped microcentrifuge tube. The hemin and HSA solutions were mixed to give a hemin:HSA molar ratio of 1.1 to 1.0, and incubated with rotation in the dark at room temperature for at least 12 hours. The resulting complex was then concentrated using a 10 kDa molecular weight cut-off ultrafiltration device (Vivaspin, Millipore), and washed in repeated cycles of concentration and dilution with 50 mM potassium phosphate buffer, pH 7.0 to reduce the final concentration of DMSO < 0.1% (v/v). Myristic acid was freshly dispersed with the aid of mild heating in 20 mM potassium phosphate pH 7.0 to obtain a suspension with a concentration of 2.5 mM. The HSA-hemin complex was incubated with myristic acid, at a fatty acid:HSA molar ratio of 4:1, by rotation at room temperature for one hour. The complex was then concentrated – in a single step – in a Vivaspin ultrafiltration device and analysed on a polyacrylamide gel to estimate the protein concentration.
Crystals were grown by sitting-drop vapour diffusion at 4°C using methods similar to those described previously [8, 9, 27]. Crystallization screens were set up at a protein concentration of >90 mg/ml using 20 to 40% PEG 3350 (Sigma-Aldrich) in 50 mM potassium phosphate buffer, pH 7.0 as the precipitant; the sitting drop initially contained 3.5 μl of the protein solution and 3.5 μl of the reservoir mixture. Three days after streak-seeding, crystals were observed at 24, 26, 28 and 30% PEG; the largest crystals were observed at 24 and 26 % PEG.
X-ray diffraction data were collected at Daresbury SRS (station 9.6) and EMBL/DESY Hamburg (beamline X11) using crystals mounted in sealed glass capillaries maintained at room temperature. The data were indexed and measured with MOSFLM and scaled with SCALA . The crystals belong to space-group P21212. Efforts to solve the phase problem by molecular replacement with AMoRe  produced a promising solution for domain II of the protein (residues 197–383) taken from the HSA-myristate structure (PDB ID 1e7g) ; however, orientations for domains I and III were not found. Since the solution for domain II appeared to yield a sensible packing arrangement for HSA molecules in the unit cell, it was used as a starting point for rigid-body refinement using CNS . The HSA-myristate model (1e7g) was initially refined as a single rigid body using data from 10–30 Å; thereafter, the protein was split into three domains (5–196, 197–383, 384–584) which were allowed to move independently and rigid body refinement was applied as the high resolution limit of the incorporated data was extended in 1 Å steps from 10 Å to 4 Å and finally to 3.2 Å. During this process, the Rfree (defined in table 1) dropped from 51.3% to 34.5%. An electron density map calculated at this stage showed clear electron density for the whole protein and the ligand molecules. Manual building in O  was interspersed with rounds of positional and group temperature factor refinement. Data collection and refinement statistics are summarized in Table 1.
We thank Delta Biotechnology (UK) for providing purified recombinant HSA (Recombumin®). We are indebted to the staff at Daresbury SRS (station 9.6) and EMBL/DESY Hamburg (X11) for assistance with data collection. JG is grateful for the award of an MRC studentship. This work was funded with grant support from the BBSRC (SC) and partially supported by the Health Science Research Grants (Research on Pharmaceutical and Medical Safety) for the MHLW.
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