Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain. Its physiological and pharmacological properties have been extensively studied over several decades Fehske et al. The most important physiological role of the protein is therefore thought to be to bring such solutes in the bloodstream to their target organs, as well as to maintain the pH and osmotic pressure of plasma.
In addition to its ordinary clinical applications, such as hypovolemic shock treatment, many investigators have attempted to utilize HSA as a carrier to deliver various drugs to their specific targets Yapel, ; Fiume et al. The primary sequence of HSA shows that the protein is a single polypeptide with residues containing 17 pairs of disulfide bridges and one free cysteine Dugiaczyk et al.
Human serum albumin, as well as serum albumin from other species, has been found to consist of three homologous domains probably derived through gene multiplication Brown, Despite many investigations using hydrodynamics, small-angle X-ray diffraction, electron microscopy and structural prediction, its three-dimensional molecular structure has remained largely unknown.
The first crystal structure of HSA at low resolution was reported by Carter and co-workers in Carter et al. The research group has also succeeded through extensive efforts in producing half a dozen different crystal forms of both HSA and non-human serum albumin, improving the resolution of their crystal structures and enhancing the knowledge of the diverse chemistry of serum albumin Ho et al. Recently, a couple of structures of HSA have been published by another research group Curry et al.
Not only did these studies shed light on the structural features of the protein, but knowledge of the three-dimensional structure also contributed to the clarification of how the protein binds various ligands. In this article, we describe a new crystal form of defatted HSA and discuss various molecular aspects of the protein as determined at the highest resolution so far reported.
A , and was used for crystallization without further purification. Crystallization took place via a conventional hanging drop procedure. Plate-shaped yellowish crystals 1. This crystal form is isomorphous to that previously reported Carter et al. Data to 3. Data collection statistics are summarized in Table I.
Isomorphous difference Patterson maps, calculated at 4. Subsequent cross difference Fourier syntheses with phases derived solely from the HgCl 2 derivative clearly showed all the heavy-atom sites for other derivatives. Initial phases at 3. Phasing statistics are listed in Table I. The initial phases were then improved and extended to 3. Sequence assignment of the atomic model was carried out using the amino acid sequence derived from the analysis of genomic DNA Minghetti et al.
Further improvement of phases were achieved using a density modification procedure in which the partial model contribution was taken into account as well as solvent flattening and histogram matching Zhang and Main, The last cycle of density modification provided an atomic model containing out of the possibly existing non-hydrogen atoms No further attempts were made to refine this model with the data from the tetragonal crystal form.
After removal of the charcoal, the solution was neutralized with 1. Elution was carried out with a linear salt gradient at NaCl concentrations of 0. Fractions corresponding to the main peak were pooled, and the entire column work was repeated until all the protein solution had been processed by the Pharmacia FPLC system.
The pooled fraction was concentrated by ultrafiltration, and dialyzed against 10 mM potassium phosphate pH 7. Crystallization was performed using a hanging drop procedure where the reservoir solution contained 50 mM potassium phosphate pH 7. A few prismatic crystals with an average size of 0. Preliminary diffraction studies revealed that crystals of both pHSA and rHSA obtained from the above procedure diffract X-rays at up to 2.
Data to a resolution of 2. Data collection statistics are summarized in Table II. A real-space rotation function at a resolution range of A subsequent translation search showed unambiguously a solution from which the relative positions of these molecules were derived. A rigid-body refinement followed by positional and overall temperature factor refinements at a resolution range of The atomic model obtained above was then subjected to a refinement procedure where simulated annealing from to K was followed by conventional positional and overall temperature factor refinements at 3.
The resulting model was manually revised using three-dimensional graphics. Hundreds of side chain atoms which had not been observed in the tetragonal crystal were identified and added to the model. All reflections within a resolution range of Although a local twofold symmetry was observed between HSA molecules in the asymmetric unit, no restraints based on this relation were applied during the refinement process.
Isolated peaks with significant height in Fo-Fc difference maps were carefully chosen as ordered water molecules if they were located near the protein molecules. Crystallographic R factor and free R factor for the current atomic model were The triclinic structure of rHSA was refined in the same way as described above, but with the refined structure of pHSA as the starting model. Current R and free R were The structural determination of HSA was performed using two different crystal forms which reveal complementary properties.
Tetragonal crystals were suitable for these experiments because of their high tolerance of the changing conditions of the mother liquor during heavy-atom derivative preparations. On the other hand, all attempts to transfer triclinic crystals from the mother liquor to any other solution failed.
This was probably due to the marked difference in packing density between the two crystal forms. After the partial molecular model had been made, the subsequent structure refinement proceeded using intensity data collected from the triclinic crystals, which diffract better up to 2. The current atomic coordinates of pHSA consist of two HSA molecules, each of which contains residues 5— non-hydrogen atoms , and seven water molecules, whereas two copies of HSA residues 5— and four water molecules are involved in the current coordinates of rHSA.
An electron density corresponding to residues 1—4 and residues — of the HSA molecule is not clearly observed, probably due to conformational flexibility at both termini. Geometrical analysis of the atomic coordinates shown in Table II indicates that bond distances, bond angles and dihedral angles were in good agreement with the ideal values proposed by Engh and Huber The peptide linkages at Pro96 adopt the cis-conformation.
A Ramachandran plot data not shown reveals that the backbone conformation for all residues falls into either energetically favorable or allowed regions Ramakrishnan and Ramachandran, ; Laskowski et al.
Average coordinate error estimated from a Luzzati analysis Luzzati, was about 0. The Fo-Fc difference map from the last refinement cycle showed no significant features attributable to mistracing or incorrect atomic coordinates. Two HSA molecules are involved in the asymmetric unit of the triclinic crystal, although HSA shows a mono-dispersive state even in condensed solutions.
The local twofold symmetry is rather strict, although no assumption was made during the refinement process regarding non-crystallographic symmetry restraints. The rotation angle to superimpose two crystallographically independent molecules in the asymmetric unit is These two independent molecules in the pHSA structure have virtually identical structures, with r. Further analysis of the residue basis clearly showed that the r.
No significant correlation was found between the residual plots of r. As can be expected from the isomorphism of crystals, the refined structures of pHSA and rHSA are virtually identical, with an r.
Fosgerau, K. Peptide therapeutics: current status and future directions. Drug Discov. Today 20 , — CAS Google Scholar. Josephson, K. Today 19 , — Morioka, T. Selection-based discovery of macrocyclic peptides for the next generation therapeutics. Duckworth, W. Insulin degradation: progress and potential. Gonser, M. Labor induction and augmentation with oxytocin: pharmacokinetic considerations.
Penchala, S. A biomimetic approach for enhancing the in vivo half-life of peptides. Kontermann, R. Half-life extended biotherapeutics. Expert Opin. Bern, M. The role of albumin receptors in regulation of albumin homeostasis: implications for drug delivery. Controlled Release , — Zaykov, A. Pursuit of a perfect insulin. Half-life extension of biopharmaceuticals using chemical methods: alternatives to PEGylation.
ChemMedChem 11 , — Kurtzhals, P. Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo. Jonassen, I. Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin.
Knudsen, L. Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration. Koehler, M. Albumin affinity tags increase peptide half-life in vivo. Dumelin, C.
A portable albumin binder from a DNA-encoded chemical library. Angew Chem. Franzini, R. Identification of structure-activity relationships from screening a structurally compact DNA-encoded chemical library.
Dennis, M. Albumin binding as a general strategy for improving the pharmacokinetics of proteins. Angelini, A. Bicyclization and tethering to albumin yields long-acting peptide antagonists. Bicyclic peptide inhibitor reveals large contact interface with a protease target.
ACS Chem. Heinis, C. Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Peters, T. Baeriswyl, V. Bicyclic peptides with optimized ring size inhibit human plasma kallikrein and its orthologues while sparing paralogous proteases.
ChemMedChem 7 , — Kenne, E. Factor XII: a drug target for safe interference with thrombosis and inflammation. A synthetic factor XIIa inhibitor blocks selectively intrinsic coagulation initiation. Wilbs, J. Improving the binding affinity of in vitro evolved cyclic peptides by inserting atoms into the macrocycle backbone. ChemBioChem 17 , — Middendorp, S. Peptide macrocycle inhibitor of coagulation factor XII with subnanomolar affinity and high target selectivity.
Markussen, J. Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs. Diabetologia 39 , — Nguyen, A. The pharmacokinetics of an albumin-binding Fab AB. Fab can be modulated as a function of affinity for albumin. Protein Eng. Download references. Alessandro Zorzi, Simon J. You can also search for this author in PubMed Google Scholar.
Correspondence to Christian Heinis. The remaining authors declare no conflict of interests. Reprints and Permissions. Zorzi, A. Nat Commun 8, Download citation. Received : 21 February Accepted : 24 May Published : 17 July Anyone you share the following link with will be able to read this content:.
Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Nature Communications Communications Biology Scientific Reports International Journal of Peptide Research and Therapeutics By submitting a comment you agree to abide by our Terms and Community Guidelines.
If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Drug delivery Peptide delivery Peptides. Abstract The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives.
Introduction Peptides combine a range of favourable qualities such as suitable affinity and selectivity, low inherent toxicity, and access to chemical synthesis which make them an attractive modality for therapeutic development 1.
Their preparation in diastereomerically pure form is a frequent target in organic synthesis, and has been achieved through several approaches, including biomimetic and organocatalytic methods. Complete conversion of the substrate to anti -diol 4 takes place without loss of selectivity and the anti diol can be easily recovered from the aqueous medium by simple extraction after denaturing the peptide with ethanol.
The albumin directed diketone reduction takes place in two steps: [20] the more accessible aliphatic carbonyl, which is exposed to the solvent in the albumin-diketone complex, is reduced first in a completely chemoselective fashion.
The aromatic carbonyl, buried inside the albumin binding site, is then reduced stereoselectively. The ability of GST-HSA to efficiently control the same reaction is again consistent with a significant degree of structural conservation. Control requires the recognition of the 1,3-diketone, stabilization by ionizable residues H and K native protein numbering in their neutral and protonated forms, respectively, [20] and complete command over the two reduction steps.
The aldol reaction is among the most important synthetic tools for the stereoselective formation of carbon-carbon bonds. Catalytic versions of this reaction have been proposed using metal catalysis, organocatalysis, and bio- and biomimetic catalysis. We have recently found that HSA and BSA catalyze the aldol addition of acetone to aldehyde 5 Figure 4b and other aromatic aldehydes with an enzyme-like mechanism.
The catalyzed process is three orders of magnitude faster than the uncatalyzed reaction, with Michaelis — Menten constants in the millimolar range [21].
The construct exhibited multiple turnover, allowing more than catalytic cycles and full conversion of a fold excess of aldehyde 5 to aldol 6. Experimental evidence supports the hypothesis that the HSA-catalyzed aldol reaction occurs through an enamine intermediate via a mechanism similar to that of type 1 aldolases.
Lysine reacts with acetone to produce the acetone enamine, which acts as nucleophile toward the aldehyde substrate. The conservation of aldolase activity in the peptide suggests that the peculiar pKa of this key lysine residue is not significantly altered in the shortened protein sequence. These data unveil the possibility of successfully obtaining aldolase peptides with enhanced efficiency and stereoselectivity from mutated libraries of GST-HSA Despite the fact that the CD and MS data presented in this paper are just consistent with, but not ultimately proving a correct folding of the albumin fragment, the protein has proven to retain the binding and catalytic activities of the whole albumin.
We are currently working in order to obtain the peptide alone after cleavage of GST, and to study its structure. Nevertheless, we believe that GST-HSA is a promising scaffold for the engineering of binders and catalysts with optimized properties and may provide an alternative to conventional antibodies as library templates for phage display.
Efavirenz was synthesized as previously reported [43]. Phosphate buffer 10 mM Na 2 PO 4 , 1. Efavirenz was first reacted with chloroacetic acid to obtain its N-carboxymethyl derivative [44]. This compound was then coupled with N-Boc-1,5-diaminopentane [45] to give tert -butyl 2- R chloro 2-cyclopropylethynyl trifluoromethyl oxo-2 H -benzo[ d ] [1] , [3] oxazin-1 4 H -yl acetamido pentylcarbamate: the acid analogue of efavirenz mg, 0.
HOBt 69 mg, 0. The solvents were removed under vacuum. All cloning was verified by sequencing. Colonies harbouring the plasmid were grown, and GST-HSA was expressed and purified as previously reported [46] with slight modification. Briefly, cells were grown to an OD of 0. Bacteria were harvested by centrifugation, and the cell pellets were lysed with 10 ml of lysis solution for each gram of bacteria, containing lysis buffer 20 mM Tris pH 8. CD spectra were obtained on a Jasco J spectropolarimeter.
Spectra were recorded in a 0. Data were collected with a data pitch of 0. Each spectrum was the average of 10 scans.
Warfarin and efavirenz stock solutions were prepared in acetonitrile. After the addition of each ligand, the fluorescence intensity at the maximum emission wavelength and the drift of such maxima were measured after equilibrium had been reached 15 min.
Excess active esters were deactivated with ethanolamine. The reference data from a deactivated flow cell were subtracted from the sensorgrams, and the interaction equilibrium constant was calculated using the BIACORE X evaluation software.
A flow rate of 0. A solution 0. Next, the mixture was filtered with a 0. Conceived and designed the experiments: F. Benedetti AT RS. Wrote the paper: F. Berti DS. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract We have identified a amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. Introduction In recent years, a number of different binding proteins have been proposed as alternatives to conventional antibody-based technologies.
Download: PPT. Figure 1. Structure of the human serum albumin HSA fragment. Chemical Reactivity Control: Diketone Reduction 1,3-Diols are naturally occurring compounds and important synthetic intermediates.
Chemical Reactivity Control: Aldolase Activity The aldol reaction is among the most important synthetic tools for the stereoselective formation of carbon-carbon bonds. Conclusion Despite the fact that the CD and MS data presented in this paper are just consistent with, but not ultimately proving a correct folding of the albumin fragment, the protein has proven to retain the binding and catalytic activities of the whole albumin.
Synthesis of the Efavirenz Derivative 2b Efavirenz was first reacted with chloroacetic acid to obtain its N-carboxymethyl derivative [44]. Fluorescence Warfarin and efavirenz stock solutions were prepared in acetonitrile.
Reduction of Diketone 3 A solution 0. Supporting Information. Figure S1. Figure S2. Figure S3. Table S1.
0コメント