pH-dependent structural change of reduced spinach plastocyanin studied by perturbed angular correlation of γ-rays and dynamic light scattering

In this study the pH-dependent structural changes of reduced spinach plastocyanin were investigated using perturbed angular correlation (PAC) of γ-rays and dynamic light scattering (DLS). PAC data of Ag-substituted plastocyanin indicated that the coordinating ligands are two histidine residues (His37, His87) and a cysteine residue (Cys84) in a planar configuration, whereas the methionine (Met92) found perpendicular to this plane is not a coordinating ligand at neutral pH. Two slightly different conformations with differences in the Cys–metal ion–His angles could be observed with PAC spectroscopy. At pH 5.3 a third coordination geometry appears which can be explained as the absence of the His87 residue and the coordination of Met92 as a ligand. With DLS the aggregation of reduced plastocyanin could be observed below pH 5.3, indicating that not only the metal binding site but also the aggregation properties of the protein change upon pH reduction. Both the structural changes at the metal binding site and the aggregation are shown to be reversible. These results support the hypothesis that the pH of the thylakoid lumen has to remain moderate during steady-state photosynthesis and indicate that low pH induced aggregation of plastocyanin might serve as a regulatory switch for photosynthesis.     

Functional and computational studies of the ligand-associated metal binding site of beta3 integrins

A combination of experimental and computational approaches was used to provide a structural context for the role of the beta3 integrin subunit ligand-associated metal binding site (LIMBS) in the binding of physiological ligands to beta3 integrins. Specifically, we have carried out (1) adhesion assays on cells expressing normal alphaIIbeta3, normal alphaVbeta3, or the corresponding beta3 D217A LIMBS mutants; and (2) equilibrium and nonequilibrium (steered) molecular dynamics (MD) simulations of eptifibatide in complex with either a fully hydrated normal alphaIIbeta3 integrin fragment (alphaIIb beta-propeller and the beta3 betaA (I-like), hybrid, and PSI domains) or the equivalent beta3 D217A mutant. Normal alphaIIbeta3 expressing cells adhered to immobilized fibrinogen and echistatin, whereas cells expressing the alphaIIbeta3 D217A LIMBS mutant failed to adhere to either ligand. Similarly, the equivalent alphaVbeta3 mutant was unable to support adhesion to vitronectin or fibrinogen. The alphaIIbeta3 D217A mutation increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the beta3 PSI domain, indicating that this mutation induced allosteric changes in the protein. Steered MD simulating the unbinding of eptifibatide from either normal alphaIIbeta3 or the equivalent beta3 D217A mutant suggested that the reduction in ligand binding caused by the LIMBS mutant required the loss of both the LIMBS and the metal ion-dependent adhesion site (MIDAS) metal ions. Our computational results indicate that the LIMBS plays a crucial role in ligand binding to alphaIIbeta3 by virtue of its effects on the coordination of the MIDAS.     

Coordination geometry for cadmium in the catalytic zinc site of horse liver alcohol dehydrogenase: studies by PAC spectroscopy

Active site substituted Cd(II) horse liver alcohol dehydrogenase has been studied by Perturbed Angular Correlation of Gamma rays Spectroscopy during turnover conditions for benzaldehyde and 4-trans-(N,N-dimethylamino)cinnamaldehyde. The ternary complex between alcohol dehydrogenase NAD⁺ and Cl^–, and the binary complex between alcohol dehydrogenase and orthophenanthroline have also been studied. The Nuclear Quadrupole Interaction parameters have been interpreted in terms of different coordination geometries for Cd(II) in the catalytic zinc site of the enzyme. Calculation of the nuclear quadrupole interaction for cadmium in the catalytic site of the enzyme with and without coenzyme, based upon the four coordinated geometries determined from X-ray diffraction, agrees with the experimentally determined values. The ternary complexes between enzyme, NAD⁺ and either Cl^– or trifluoroethanol and the binary complex between enzyme and orthophenanthroline have almost identical spectral parameters which are not consistent with a four coordinated geometry, but are consistent with a five coordinated geometry. The nonprotein ligands for the ternary complex with trifluoroethanol are suggested to be an alkoxide group and a water molecule. The Nuclear Quadrupole Interaction parameters for the productive ternary complex between enzyme, NADH and an aldehyde is consistent with the four coordinated geometry predicted from X-ray diffraction data having the carbonyl group of the aldehyde substituting the water molecule as ligand to the metal.Abbreviations LADH Horse liver alcohol dehydrogenase - H₄Zn₂LADH derivative of LADH free of zinc in the catalytic site - ¹¹¹CdZn₂LADH derivative of LADH with ¹¹¹Cd (carrier free) in the catalytic site - Cd₂Zn₂LADH derivative of LDH with 2 mole of Cd(II) per mole LADH in the catalytic site - PAC pertubed angular correlation of gamma rays - NQI Nuclear quadrupole interaction - AOM Angular overlap model - trifluoroethanol 2,2,2-trifluoroethanol - DACA trans-4-(N,N-dimethylamino)cinnamaldehyde - NAD⁺ and NADH oxidized and reduced nicotinamide adenine dinucleotide - NADH₂ reduced 1,4,5,6-tetrahydronicotinamide adenine dinucleotide The experimental work was carried out at the Niels Bohr Institute Risø, 4000 Roskilde and Blegdamsvej 19, 2100 Copenhagen, Denmark Offprint requests to: R. Bauer     

Conformational change of erythroid alpha-spectrin at the tetramerization site upon binding beta-spectrin

We previously determined the solution structures of the first 156 residues of human erythroid alpha-spectrin (SpalphaI-1-156, or simply Spalpha). Spalpha consists of the tetramerization site of alpha-spectrin and associates with a model beta-spectrin protein (Spbeta) with an affinity similar to that of native alpha- and beta-spectrin. Upon alphabeta-complex formation, our previous results indicate that there is an increase in helicity in the complex, suggesting conformational change in either Spalpha or Spbeta or in both. We have now used isothermal titration calorimetry, circular dichroism, static and dynamic light scattering, and solution NMR methods to investigate properties of the complex as well as the conformation of Spalpha in the complex. The results reveal a highly asymmetric complex, with a Perrin shape parameter of 1.23, which could correspond to a prolate ellipsoid with a major axis of about five and a minor axis of about one. We identified 12 residues, five prior to and seven following the partial domain helix in Spalpha that moved freely relative to the structural domain in the absence of Spbeta but when in the complex moved with a mobility similar to that of the structural domain. Thus, it appears that the association with Spbeta induced an unstructured-to-helical conformational transition in these residues to produce a rigid and asymmetric complex. Our findings may provide insight toward understanding different association affinities of alphabeta-spectrin at the tetramerization site for erythroid and non-erythroid spectrin and a possible mechanism to understand some of the clinical mutations, such as L49F of alpha-spectrin, which occur outside the functional partial domain region.     

The posttranslational modification of β-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site

Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of β-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus β-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of β-endorphin indicated that the posttranslational processing of β-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.     

The atypical iron-coordination geometry of cytochrome f remains unchanged upon binding to plastocyanin, as inferred by XAS

The transient complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by X-ray Absorption Spectroscopy in solution, using both proteins in their oxidized and reduced states. Fe K-edge data mainly shows that the atypical metal coordination geometry of cytochrome f, in which the N-terminal amino acid acts as an axial ligand of the heme group, remains unaltered upon binding to its redox partner, plastocyanin. This fact suggests that cytochrome f provides a stable binding site for plastocyanin and minimizes the reorganization energy required in the transient complex formation, which could facilitate the electron transfer between the two redox partners.     

Role of beta-lactam carboxyl group on binding of penicillins and cephalosporins to class C beta-lactamases

Molecular models for the Henry Michaelis complexes of Enterobacter cloacae, a class C beta-lactamase, with penicillin G and cephalotin have been constructed by using molecular mechanic calculations, based on the AMBER force field, to examine the molecular differentiation mechanisms between cephalosporins and penicillins in beta-lactamases. Ser318Ala and Thr316Ala mutations in both complexes and Asn346Ala and Thr316Ala/Asn346Ala double mutation in penicillin G complex have also been studied. Results confirm that Thr316, Ser318, and Asn346 play a crucial role in the substrate recognition, via their interactions with one of the oxygens of the antibiotic carboxyl group. Both mutation Ser318Ala and Thr316Ala strongly affect the correct binding of cephalotin to P99, the first mainly by precluding the discriminating salt bridge between carboxyl and serine OH groups, and the second one by the Ser318, Lys315, and Tyr150 spatial rearrangements. On the other hand, Ser318Ala mutation has little effect on penicillin G binding, but the Thr316Ala/Asn346Ala double mutation causes the departure of the antibiotic from the oxyanion hole. Molecular dynamic simulations allow us to interpret the experimental results of some class C and A beta-lactamases.     

Carbohydrate binding sites in a pancreatic alpha-amylase-substrate complex, derived from X-ray structure analysis at 2.1 A resolution.

The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duée E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA.     

Anatomy of a conformational transition of beta-strand 6 in soybean beta-amylase caused by substrate (or inhibitor) binding to the catalytical site.

A computational study of the five soybean beta-amylase X-ray structure reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the beta-strand 6 (residues 341-343) in the (beta/alpha)8 molecular scaffold. Backbone distortions that involve considerable changes in the phi and psi angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA-layer (at the C-terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand-binding process, there is a negative enthalpy change for these residues (-27 and -34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed "stability-function" hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452-456). They are also in good agreement with other experimental results in the literature that describe the role of the 341-343 segment in beta-amylase activity. Site-directed mutagenesis focused on these residues could be useful for undertaking functional studies of beta-amylase.     

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc-beta-lactamase

Beta-lactamases are involved in bacterial resistance. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn-beta-lactamase X-ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in beta-lactam hydrolysis, the replacement by Ser of the zinc-liganding residue Cys168 in the Zn-beta-lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono-Zn form, but active in the di-Zn form. The structure of the mono-Zn form of the C168S mutant has been determined at 1.85 A resolution. Ser168 occupies the same position as Cys168 in the wild-type enzyme. The protein residues mostly affected by the mutation are Asp90-Arg91 and His210. A critical factor for the activity of the mono-Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs catalysis. The evolution of a large superfamily including Zn-beta-lactamases suggests that they may not all share the same mechanism.     

Hierarchy of ADAM12 binding to integrins in tumor cells

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADAM12. However, when alpha9beta1 integrin is not expressed--as in many carcinoma cells--other members of the beta1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported alpha9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via alpha9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an alpha9 integrin-dependent manner also attached to ADAM12 in which the putative alpha9beta1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate beta1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the alpha9beta1 integrin was Mn(2+)-independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the alpha9beta1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating alpha9beta1 integrin cell spreading activity in response to ADAM12.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

The complexed structure and antimicrobial activity of a non-beta-lactam inhibitor of AmpC beta-lactamase

Beta-lactamases are the major resistance mechanism to beta-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to beta-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-beta-lactam inhibitors of beta-lactamases were investigated for complementarity to the structure of AmpC beta-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo(b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a Ki of 27 nM. This inhibitor is chemically dissimilar to beta-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 A resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-beta-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against beta-lactams in conjunction with beta-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many beta-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-beta-lactam inhibitors.     

Induced chirality upon binding of cis-parinaric acid to bovine beta-lactoglobulin: spectroscopic characterization of the complex

Binding of the polyunsaturated cis-parinaric acid to bovine β-lactoglobulin (BLG) was studied by circular dichroism (CD), electronic absorption spectroscopy and mass spectrometry methods. Upon protein binding, the UV absorption band of parinaric acid is red shifted by ca. 5 nm, showing hypochromism and reduced vibrational fine structure, suggesting that the ligand binds as a monomer in non-planar geometry. In the CD spectra measured at pH 7.36 and 8.5 a strong, negative Cotton band appears centered at 310 nm (Δε=−25 M⁻¹ cm⁻¹) corresponding to the long-wavelength absorption band of cis-parinaric acid. The source of this induced optical activity is the helical distortion of the polyene chromophore caused by the chiral protein environment. From CD spectral data the value of the association constant was calculated to be 4.7×10⁵ M⁻¹ at pH 7.36. CD and mass spectrometry measurements showed that parinaric acid binds weakly to BLG in acidic solution, though small peaks at mass 18559 and 18645 can be obtained in the reconstructed electrospray mass spectrum; these correspond to the binding of parinaric acid in 1:1 stoichiometry to both monomer variants of BLG B and A. The hydrophobic interior cavity of BLG was assigned as the primary binding site of cis-parinaric acid.     

The co-crystal structure of unliganded bovine α-thrombin and prethrombin-2: Movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding

Unliganded bovine α-thrombin and prethrombin-2 have been co-crystallized, in space group P2₁2₁2, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 Å and 2.3 Å, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (R_free = 0.255) and 0.190 (R_free = 0.282) for the salt and PEG2K models, respectively. The apo-enzyme form of bovine α-thrombin shows dramatic shifts in placement for the Tyr-Pro-Pro-Trp segment, for Glu-192, and for the catalytic residues His-57 and Ser-195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7-19 of fibrinogen Aa with a non-cleavable scissile bond), (2) enzyme-inhibitor complex (D-Phe-Pro-Arg chloromethylketone), (3) enzyme product complex (residues 7-16 of fibrinopeptide A), and (4) the exosite complex (residues 53-64 of hirudin). The structures of bovine and human prethrombin-2 are generally similar to one another (RMS deviation of 0.68 8,) but differ significantly in the Arg-15/Ile-16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin-2, analogous to that seen for trypsinogen.     

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.     

Crystal structure of Drosophila angiotensin I-converting enzyme bound to captopril and lisinopril

This study provides evidence that treatment with preclustered ephrin A5-Fc results in a substantial increase in the stability of the p110γ PI-3 kinase associated with EphA8, thereby enhancing PI-3 kinase activity and cell migration on a fibronectin substrate. In contrast, co-expression of a lipid kinase-inactive p110γ mutant together with EphA8 inhibits ligand-stimulated PI-3 kinase activity and cell migration on a fibronectin substrate, suggesting that the mutant has a dominant negative effect against the endogenous p110γ PI-3 kinase. Significantly, the tyrosine kinase activity of EphA8 is not important for either of these processes. Taken together, our results demonstrate that the stimulation of cell migration on a fibronectin substrate by the EphA8 receptor depends on the p110γ PI-3 kinase but is independent of a tyrosine kinase activity.     

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.