Subdomains IIB Research Articles

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

Syntheses, crystal structures of two Fe(III) Schiff base complexes with chelating o-vanillin aroylhydrazone and exploration of their bio-relevant activities

Two novel Fe(III) complexes, Fe(HL1)2Cl·1.25H2O (1) and Fe(HL2)2·Et3NH·H2O (2) (H2L1 = o-vanillin benzoylhydrazone, H3L2 = o-vanillin salicylhydrazone) are prepared. X-ray single crystal diffraction confirms that the hydrazone ligands can be chelated to iron centre resulting in a six-coordinate octahedral configuration. Both complexes show major intercalation effect to the herring sperm deoxyribonucleic acid (HS-DNA) with high binding constants of 2.01 × 104 M−1 and 2.24 × 104 M−1, respectively. Molecular docking studies reveal both complexes can intercalate at the gap of DC5-DG2 and DG6-DC1 base pairs of DNA hexamer (1Z3F). The interaction of the complex 1 with plasmid pBR322 DNA induces distinguishable alterations of the DNA morphology. Further, the structure of plasmid pBR322 DNA treated with complex 1 in the presence of ascorbic acid has been damaged probably due to the reactive oxygen species (ROS) generation. What's more, both complexes show high affinity with bovine serum albumin (BSA), the binding constants measured by fluorescence techniques are 5.75 × 106 M−1 and 4.39 × 107 M−1, respectively. Molecular docking demonstrates that the complexes prefer the binding pocket of site III (subdomain IIB) of BSA (PDB ID: 4F5S). Similarly, dynamic light scattering (DLS) reveals that the complexes not only bind to BSA but also induce bigger size aggregates as the concentration increases.

Crystal structure of a cross‐species reactive nanobody in complex with human serum albumin

Nanobodies are single‐domain antibodies derived from the unique heavy‐chain antibodies found in camelid species. Nanobodies are attractive therapeutics as they are easy to produce, are highly stable, and can potentially bind targets inaccessible to traditional antibody formats. However, due to their small molecular weight, one disadvantage associated with nanobodies is rapid renal clearance resulting in a short serum half‐life. One innovative solution to increasing the serum half‐life of nanobodies is to make use of bispecific nanobodies. In this format, a therapeutic nanobody is fused to a second nanobody specific for the highly abundant plasma protein, human serum albumin. Such bispecific nanobody constructs targeting serum albumins have been shown to increase the half‐life of therapeutic nanobodies. The use of an albumin specific nanobody that cross reacts with albumin found in commonly used research animals is particularly desirable as the bispecific nanobody could be used pre‐clinical animal trials. Previously, we have generated a panel nanobodies which cross‐react with the serum albumins of humans, macaques, mouse, and rat, through immunization of a llama with human serum albumin. To determine the basis of the cross‐species albumin reactivity of the nanobodies we have determined the X‐ray structure of the nanobody M79 in complex with human serum albumin. M79 is cross reactive with serum albumins from macaques, mouse, and rat. The structure revealed of the three nanobody binding loops, only two of the loops (CDR2 and CDR3) interacted with human serum albumin. The M79 nanobody bound to the apex of subdomain IIB on human serum albumin, specifically forming polar contacts with residues D308, D314, N318, and E321. Comparison of the serum albumin sequences from human, macaques, mouse, and rat show that the residues in serum albumin that bound M79 are highly conserved amongst the species explaining the nanobodies’ cross‐reactivity. These results give insights on the cross‐species specificity of M79 towards serum albumin, and can bolster the use of M79 in animal model experiments.

Exploring the binding interaction between bovine serum albumin and perindopril as well as influence of metal ions using multi-spectroscopic, molecular docking and DFT calculation

The binding characteristics of BSA with perindopril, a third-generation angiotensin-converting enzyme inhibitor, was investigated using various spectroscopic approaches combined with molecular simulation including molecular docking and density functional theory calculation. Meantime, the influence of some trace metal ions on the binding characteristics of BSA with perindopril was investigated. The findings indicated the perindopril quenched BSA fluorescence in a static way. The values of binding site (n) and binding constant (Kb) of perindopril on BSA were close to 1 and 8.12 × 103 M−1 at 298 K, respectively, indicating that the binding affinity was moderate and the stoichiometric ratio of the perindopril-BSA complex was 1:1. Thermodynamic analysis results revealed that the ΔG0, ΔH0, and ΔS0 values were negative, demonstrating the binding interaction was a spontaneous and exothermic process, the interaction forces were mainly hydrogen bonds and van der Waals forces. The results of the replacement experiments and molecular simulations indicated that perindopril bound to the groove between sub-domain IIB and IIA. Furthermore, the results also revealed the BSA conformation altered slightly during binding with perindopril and the existence of transition metal ions would result in the increase in the Kb value of perindopril on BSA, resulting in the decline in the concentration of free perindopril. The frontier molecular orbitals and other quantum chemical parameters as well as conformation of perindopril in BSA complex obviously altered for forming the more stable perindopril-BSA complex.

Investigation of new indole derivatives of bovine serum albumin using spectroscopic and molecular docking techniques

ABSTRACTWe investigated the interaction of two derivatives of bis (indolyl) methane with bovine serum albumin (BSA) using spectroscopic and molecular docking calculations. Fluorescence quenching measurements revealed that the quenching mechanism was static. Fӧrster energy transfer measurements, synchronous fluorescence spectroscopy and docking studies demonstrated that both bis(indolyl)methanes bound to the Trp residues of BSA. The docking study confirmed that both bis(indolyl)methanes form hydrogen bonds and van der Waals interactions with BSA. Our molecular docking study indicated that the compounds are located within the binding pocket of subdomains IIB and IB of BSA. Fourier transform infrared spectroscopy demonstrated that both bis(indolyl)methane derivatives can interact with BSA and can affect the secondary structure of BSA.

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4-Å resolution

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.

NMR structure and dynamics of Q4D059, a kinetoplastid-specific and conserved protein from Trypanosoma cruzi.

Q4D059 (UniProt accession number), is an 86-residue protein from Trypanosoma cruzi, conserved in the related kinetoplastid parasites Trypanosoma brucei and Leishmania major. These pathogens are the causal agents of the neglected diseases: Chagas, sleeping sickness and leishmaniases respectively and had recently their genomes sequenced. Q4D059 shows low sequence similarity with mammal proteins and because of its essentiality demonstrated in T. brucei, it is a potential target for anti-parasitic drugs. The 11 hypothetical proteins homologous to Q4D059 are all uncharacterized proteins of unknown function. Here, the solution structure of Q4D059 was solved by NMR and its backbone dynamics was characterized by (15)N relaxation parameters. The structure is composed by a parallel/anti-parallel three-stranded β-sheet packed against four helical regions. The structure is well defined by ca. 9 NOEs per residue and a backbone rmsd of 0.50±0.05 Å for the representative ensemble of 20 lowest-energy structures. The structure is overall rigid except for N-terminal residues A(9) to D(11) at the beginning of β1, K(38), V(39) at the end of helix H3 with rapid motion in the ps-ns timescale and G(25) (helix H2), I(68) (β2) and V(78) (loop 3) undergoing internal motion in the μs-ms timescale. Limited structural similarities were found in protein structures deposited in the PDB, therefore functional inferences based on protein structure information are not clear. Q4D059 adopts a α/β fold that is slightly similar to the ATPase sub-domain IIB of the heat-shock protein 70 (HSP70) and to the N-terminal domain of the ribosomal protein L11.

Interplay between E. coli DnaK, ClpB and GrpE during Protein Disaggregation

The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: Escherichia coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the middle domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomains IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK–ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.

ATPase subdomain IA is a mediator of interdomain allostery in Hsp70 molecular chaperones.

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.

Interaction Characteristics with Bovine Serum Albumin and Retarded Nitric Oxide Release of ZCVI4-2, a New Nitric Oxide-Releasing Derivative of Oleanolic Acid

ZCVI₄-2 is a newly developed furoxan-based nitric oxide-releasing derivative of oleanolic acid. It exhibited strong cytotoxicity against human hepatocellular carcinoma (HCC) in vitro and significantly inhibited the growth of HCC tumors in vivo. However, its low aqueous solubility and toxicity due to the fast release of nitric oxide (NO) in blood challenged its formulation. In the present investigation, the interaction characteristics of ZCVI₄-2 with bovine serum albumin (BSA) were studied by fluorescence spectrometry, synchronous fluorescence spectra and Fourier transform-infrared (FT-IR). It was found that ZCVI₄-2 concentration, temperature and pH had significant effect on the interactions. ZCVI₄-2 was able to bind BSA with high affinity, low temperature and neutral pH favor the binding. The interaction exhibited to be a spontaneous and exothermic process. ZCVI₄-2 was buried in the hydrophobic pocket in subdomain IIB of BSA and the exact binding site was around 3.83 nm in average from Trp²¹². The NO releasing characteristics of nanocomplexes were compared with ZCVI₄-2 solution by Griess Reagent Method. It was found that the release of NO from ZCVI₄-2/BSA nanocomplexes was retarded significantly, thus making ZCVI₄-2 into a BSA-bound nanocomplexes had the great potential to lower the toxicity due to the absence of organic solvents and surfactants and meanwhile the sustained release of NO.

Direct inter-subdomain interactions switch between the closed and open forms of the Hsp70 nucleotide-binding domain in the nucleotide-free state

The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the ;closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA

The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus genomic RNA, is an attractive target for antiviral drugs. Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca 2+-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca 2+. Ca 2+-dependent p94 mutants were screened using “p94-trapping”, which is an application of yeast genetic reporter system called “proteinase-trapping”. Several amino acids were revealed as critical for apparent Ca 2+-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.

Electrostatic interactions of domain III stabilize the inactive conformation of mu-calpain.

The ubiquitous mu- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human mu-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive micro-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.

Immobilised cooper(II) ion-affinity chromatography of natural mutants of human serum albumin and proalbumin

Interaction of natural mutants of human serum albumin and proalbumin with chelated and immobilised Cu(II) ions (IDA-Cu(II)) has been studied by chromatography. All the studied variants bound to IDA-Cu(II) columns under the experimental conditions employed (pH 6.8; 0.5M NaCl) and were displaced by affinity elution with imidazole. Variants with a propeptide, i.e. the proalbumins (proAlb) Varese, Blenheim, and Kaikoura, and those with an extra Arg residue (Arg-1) at the N-terminus, i.e. the albumins (Alb) Redhill and Arg-Alb, had the highest affinities for immobilised Cu2+. Binding to IDA-Cu(II) was moderately increased by modification of the C-terminal region of the albumin molecule (i.e. Albs Venezia and Catania) but was not affected by structural changes at the N-terminal end (i.e. Alb Blenheim) and in subdomains IIB (i.e. Albs Herborn and French Reading) and IIIB of the molecule (i.e. Alb Casebrook). The different metal-binding of these variants was further confirmed in other studies using IDA-Cu(II) chromatography with ammonium chloride as eluent. The variant Arg-Alb was displaced from IDA-Cu(II) only on protracted irrigation of the columns with ammonium chloride (1 M) whereas the three proalbumins studied could not be displaced. All the other albumin variants were readily eluted from the columns with ammonium chloride (1 M), although their retention behaviur varied. Analysis of the (pro)albumin variants for Cu2+ content, after their passage through IDA-Cu(II) columns, showed that these proteins differed in their ability to scavenge Cu2+ from its immobilised chelate, IDA-Cu(II). The different scavenging capabilities seem to be related to different affinities of these variants with regard to primary binding of free Cu2+ ions. A molecular explanation of the different IDA-Cu(II) binding of the tested (pro)albumins is proposed; this makes use of the possibility given by available views of the crystal structure of human serum albumin. More practically, the possibility of resolving some of the tested (pro)albumin variants from their normal counterpart (endogenous albumin) by IDA-Cu(II) chromatography could aid the separation and further purification of these variants.

Structural basis for possible calcium-induced activation mechanisms of calpains.

The calpains form a growing family of structurally related intracellular multidomainal cysteine proteinases, which exhibit a catalytic domain distantly related to papain. In contrast to papain, however, their activity in most cases depends on calcium. The calpains are believed to play important roles in cytoskeletal remodeling processes, cell differentiation, apoptosis and signal transduction, but have also been implicated in muscular dystrophy, ischemia, traumatic brain injury, neurodegenerative diseases, rheumatoid arthritis and cataract formation. The best characterized calpains are the ubiquitously expressed mu- and m-calpains, consisting of a common 30 kDa small S-subunit (domains V and VI) and slightly differing 80 kDa large L-subunits (domains I to IV). We have recently determined the 2.3 A structure of recombinant full-length human m-calpain in the absence of calcium, which reveals that the catalytic domain and the two calmodulin-like domains, previously believed to represent the unique calcium switch, are not positioned adjacent to each other, but are separated by the beta-sandwich domain III, which distantly resembles C2 domains. Although the catalytic domain of apocalpain is strongly disrupted compared to papain (which explains its inactivity in the absence of calcium), the crystal structure reveals several sites where calcium could bind, thereby causing a subdomain fusion to form a papain-like catalytic center. All current evidence points to the cooperative interaction of several calcium binding sites. Sites identified include the three EF-hand binding sites in each calmodulin-like domain, the negatively charged segments arranged around the active-site cleft (provided by both catalytic subdomains), as well as an exposed acidic loop of domain III, whose charge compensation could allow the adjacent barrel-like subdomain IIb to move toward the helical subdomain IIa. The Gly-rich S-chain N-terminus and the calcium-loaded acidic loop could target the conventional calpains to cellular/nuclear membranes, thereby explaining their strongly reduced calcium requirement in vivo and in vitro in the presence of acidic phospholipids.

Five recombinant fragments of human serum albumin—tools for the characterization of the warfarin binding site

Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.

Structural characterization of three genetic variants of human serum albumin modified in subdomains IIB and IIIA.

Three new genetic variants of human serum albumin have been detected in Italy by routine clinical electrophoresis. Albumin Milano Slow is common in Northern Italy, while albumins Liprizzi and Trieste, which are fast migrating, are rare and local variants. Isoelectric focusing analysis of the CNBr fragments obtained from the carboxymethylated alloalbumins in all cases localized the mutation to fragment CB5 (residues 330-446). The modified CNBr fragments were isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptides revealed that all the variants are caused by single point mutations: Trieste, Lys359-->Asn, Milano Slow, Asp375-->His, and Liprizzi, Arg410-->Cys. These results were confirmed by sequence determination of a variant V8 peptide in the case of Trieste, and by DNA sequence analysis for the other two variants. The DNA analysis showed a G-->C transversion at nucleotide position 11969 for albumin Milano Slow, and a C-->T transition at position 13251 for Liprizzi. The latter represents a mutation at a hypermutable CpG dinucleotide site. Albumins Trieste and Milano Slow, as most of the variants thus far described, have mutations involving residues on the surface of the molecule. In contrast, albumin Liprizzi represents the first example of a mutation in the most active binding pocket of the molecule, placed in subdomain IIIA.