Structure, specificity, and mode of interaction for bacterial albumin-binding modules.

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.     

Crystal structure and biological implications of a bacterial albumin binding module in complex with human serum albumin

Many bactericide species express surface proteins that interact with human serum albumin (HSA). Protein PAB from the anaerobic bacterium Finegoldia magna (formerly Peptostreptococcus magnus) represents one of these proteins. Protein PAB contains a domain of 53 amino acid residues known as the GA module. GA homologs are also found in protein G of group C and G streptococci. Here we report the crystal structure of HSA in complex with the GA module of protein PAB. The model of the complex was refined to a resolution of 2.7 A and reveals a novel binding epitope located in domain II of the albumin molecule. The GA module is composed of a left-handed three-helix bundle, and residues from the second helix and the loops surrounding it were found to be involved in HSA binding. Furthermore, the presence of HSA-bound fatty acids seems to influence HSA-GA complex formation. F. magna has a much more restricted host specificity compared with C and G streptococci, which is also reflected in the binding of different animal albumins by proteins PAB and G. The structure of the HSA-GA complex offers a molecular explanation to this unusually clear example of bacterial adaptation.     

Structure, dynamics, and stability variation in bacterial albumin binding modules: implications for species specificity

Protein G-related albumin-binding (GA) modules are frequently expressed on the surfaces of bacterial cells. The limited amino acid sequence variation among GA modules results in structural and functional differences with possible implications for bacterial pathogenesis and host specificity. In particular, the streptococcal G148-GA3 and F. magna ALB8-GA albumin-binding domains exhibit a degree of structural and dynamic diversity that may account for their varied affinities for different species of albumin. To explore the impact of GA module polymorphisms on albumin binding and specificity, we recently used offset recombinant PCR to shuffle seven artificially constructed representatives of the GA sequence space and scan the phage-displayed recombinant domains for mutations that supported binding to the phylogenetically distinct human and guinea pig serum albumins (HSA and GPSA) (Rozak et al. (2006) Biochemistry 45, 3263-3271). Surprisingly, phage selection revealed an overwhelming preference for a single recombinant domain (PSD-1, phage-selected domain-1) regardless of whether the phages were enriched for their abilities to bind one or both of these albumins. We describe here the NMR-derived structure, dynamics, and stability of unbound PSD-1. Our results demonstrate that increased flexibility is not a requirement for broadened specificity, as had been suggested earlier (Johansson et al. (2002) J. Mol. Biol. 316, 1083-1099), because PSD-1 binds the phylogenetically diverse HSA and GPSA even more tightly than G148-GA3 but is less flexible. The structural basis for albumin-binding specificity is analyzed in light of these new results.     

Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.     

An artificially evolved albumin binding module facilitates chemical shift epitope mapping of GA domain interactions with phylogenetically diverse albumins

Protein G-related albumin-binding (GA) modules occur on the surface of numerous Gram-positive bacterial pathogens and their presence may promote bacterial growth and virulence in mammalian hosts. We recently used phage display selection to evolve a GA domain, PSD-1 (phage selected domain-1), which tightly bound phylogenetically diverse albumins. With respect to PSD-1's broad albumin binding specificity, it remained unclear how the evolved binding epitope compared to those of naturally occurring GA domains and whether PSD-1's binding mode was the same for different albumins. We investigate these questions here using chemical shift perturbation measurements of PSD-1 with rabbit serum albumin (RSA) and human serum albumin (HSA) and put the results in the context of previous work on structure and dynamics of GA domains. Combined, these data provide insights into the requirements for broad binding specificity in GA-albumin interactions. Moreover, we note that using the phage-optimized PSD-1 protein significantly diminishes the effects of exchange broadening at the binding interface between GA modules and albumin, presumably through stabilization of a ligand-bound conformation. The employment of artificially evolved domains may be generally useful in NMR structural studies of other protein-protein complexes.     

G148-GA3: a streptococcal virulence module with atypical thermodynamics of folding optimally binds human serum albumin at physiological temperatures

The third albumin binding domain of streptococcal protein G strain 148 (G148-GA3) belongs to a novel class of prokaryotic albumin binding modules that is thought to support virulence in several bacterial species. Here, we characterize G148-GA3 folding and albumin binding by using differential scanning calorimetry and isothermal titration calorimetry to obtain the most complete set of thermodynamic state functions for any member of this medically significant module. When buffered at pH 7.0 the 46-amino acid alpha-helical domain melts at 72 degrees C and exhibits marginal stability (15 kJ/mol) at 37 degrees C. G148-GA3 unfolding is characterized by small contributions to entropy from non-hydrophobic forces and a low DeltaCp (1.1 kJ/(deg mol)). Isothermal titration calorimetry reveals that the domain has evolved to optimally bind human serum albumin near 37 degrees C with a binding constant of 1.4 x 10 7 M(-1). Analysis of G148-GA3 thermodynamics suggests that the domain experiences atypically small per residue changes in structural dynamics and heat capacity while transiting between folded and unfolded states.     

Predicting the binding properties of cibacron blue F3GA in affinity separation systems

The binding properties of cibacron blue F3GA (CB-F3GA) bound to a model NAD(P)H/FAD(H2)-dependent protein system, namely cytosolic quinone reductase (QR), was characterized by AMBER in an attempt to address the binding properties of immobilized CB-F3GA used in the separation of serum albumin. A favorable binding free energy of -4.52kcal/mol (KD=5.09 x 10(-4)kcal/mol) was determined for CB-F3GA binding by MM-PBSA method, which was found to be a ballpark estimate of empirical values reported in literature (DeltaG approximately -6kcal/mol). We propose that CB-F3GA primarily follows a class III binding motif in presence of FAD in the binding site of QR in solution, while a class II binding motif is observed in the crystal form. It was found that favorable van der Waals/hydrophobic interactions take place in the binding site making a major contribution to a favorably dominating enthalpy of binding (DeltaHtot=-25.87kcal/mol) as compared to a disfavorable binding entropy term (TDeltaStot=-21.35kcal/mol). Additional MM-PBSA experiments in the absence of FAD gave rise to a disfavorable binding free energy for CB in complex with QR, suggesting that FAD is an essential determinant of CB-F3GA binding. This is in contrast to an earlier observation of Denizli et al. on separation of human serum albumin (HSA) by immobilized CB-F3GA in the absence of FAD. Therefore, a class I binding model for CB-F3GA is proposed here to account for the efficient separation of HSA in affinity chromatography systems.     

The mitogenic activity of type III bacterial Ig binding proteins (protein G) for human peripheral blood lymphocytes is not related to their ability to react with human serum albumin or IgG

The mitogenic potential of bacterial IgG Fc binding proteins for human PBL is controversial. Wild type and recombinant type III IgG Fc binding proteins induce a wide spectrum of proliferative responses ranging from non-mitogenic to potent responses. To understand the reason for these differences, three recombinant forms of a type III IgG Fc binding protein derived from a single human group C streptococcal strain, 26RP66, were generated. Form I bound human IgG and human serum albumin, form II bound IgG alone and form III bound human serum albumin alone. These functionally distinct forms were compared with the corresponding wild type preparation from the same strain for mitogenic potential. A mitogenic response was induced only with the form I recombinant or the native wild type protein. These proteins shared the functional characteristics of binding human serum albumin and IgG. Mixtures of the IgG binding (form II) and human serum albumin binding fragments (form III) failed to reconstitute the mitogenic potential of the full length proteins. These results demonstrate that the type III IgG Fc binding protein has mitogenic potential for human PBL that is not related to its ability to react with human serum albumin or IgG.     

A dynamic model for bilirubin binding to human serum albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.     

Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules

The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.     

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.     

A tomato endo-beta-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49)

A critical structural feature of many microbial endo-beta-1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally thought to have the capacity to degrade crystalline cellulose. We report the biochemical characterization of a tomato EGase, Solanum lycopersicum Cel8 (SlCel9C1), with a distinct C-terminal noncatalytic module that represents a previously uncharacterized family of CBMs. In vitro binding studies demonstrated that this module indeed binds to crystalline cellulose and can similarly bind as part of a recombinant chimeric fusion protein containing an EGase catalytic domain from the bacterium Thermobifida fusca. Site-directed mutagenesis studies show that tryptophans 559 and 573 play a role in crystalline cellulose binding. The SlCel9C1 CBM, which represents a new CBM family (CBM49), is a defining feature of a new structural subclass (Class C) of plant EGases, with members present throughout the plant kingdom. In addition, the SlCel9C1 catalytic domain was shown to hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and a variety of plant cell wall polysaccharides.     

A CBM20 low-affinity starch-binding domain from glucan, water dikinase

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.     

An engineered ultra‐high affinity Fab‐Protein G pair enables a modular antibody platform with multifunctional capability

Engineered recombinant antibody‐based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab‐based affinity reagents in a “plug and play” fashion. This platform exploits the ultra‐tight binding interaction between affinity matured variants of a Fab scaffold (Fab^S) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi‐specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab‐GA1 platform, we applied it to a detection proximity assay based on the β‐lactamase (BL) split enzyme system. We also show the bi‐specific capabilities of the module by using it in context of a Bi‐specific T‐cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T‐cells to cancer cells. We show that GA1‐Fab modules are easily engineered into potent cell‐killing BiTE‐like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer‐related targets in a plug and play fashion.     

Proteinaceous factor(s) in culture supernatant fluids of bifidobacteria which prevents the binding of enterotoxigenic Escherichia coli to gangliotetraosylceramide.

We have examined the competitive binding of several species of Bifidobacterium and Escherichia coli Pb176, an enterotoxigenic E. coli (ETEC) strain, to gangliotetraosylceramide (asialo GM1 or GA1), a common bacterium-binding structure, and identified a factor(s) in the Bifidobacterium culture supernatant fluid that inhibits the binding of E. coli Pb176 to GA1. The ETEC strain we used expresses colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3. Competitive exclusion of ETEC from GA1 molecules by Bifidobacterium cells was found by an in vitro thin-layer chromatography overlay binding suppression assay. However, the ETEC cells were less effective in blocking the adherence of Bifidobacterium cells to GA1. These findings suggest that the two bacterial species recognize different binding sites on the GA1 molecule and that the mechanism of competitive exclusion is not due to specific blockage of a common binding site on the molecule. The neutralized culture supernatant fluids of Bifidobacterium species, including that of Bifidobacterium longum SBT 2928 (BL2928), showed remarkable inhibition of the ETEC binding to GA1. Our results suggest that the binding inhibitor produced by BL2928 is a proteinaceous molecule(s) with a molecular weight around or over 100,000 and a neutral isoelectric point. The binding inhibitor produced by BL2928 and other Bifidobacterium species is estimated to contribute to their normal anti-infectious activities by preventing the binding of pathogenic strains of E. coli to GA1 on the surface of the human intestinal mucosa.     

The Molecular Clock Runs at Different Rates Among Closely Related Members of a Gene Family

The serum albumin gene family is composed of four members that have arisen by a series of duplications from a common ancestor. From sequence differences between members of the gene family, we infer that a gene duplication some 580 Myr ago gave rise to the vitamin D–binding protein (DBP) gene and a second lineage, which reduplicated about 295 Myr ago to give the albumin (ALB) gene and a common precursor to α-fetoprotein (AFP) and α-albumin (ALF). This precursor itself duplicated about 250 Myr ago, giving rise to the youngest family members, AFP and ALF. It should be possible to correlate these dates with the phylogenetic distribution of members of the gene family among different species. All four genes are found in mammals, but AFP and ALF are not found in amphibia, which diverged from reptiles about 360 Myr ago, before the divergence of the AFP-ALF progenitor from albumin. Although individual family members display an approximate clock-like evolution, there are significant deviations—the rates of divergence for AFP differ by a factor of 7, the rates for ALB differ by a factor of 2.1. Since the progenitor of this gene family itself arose by triplication of a smaller gene, the rates of evolution of individual domains were also calculated and were shown to vary within and between family members. The great variation in the rates of the molecular clock raises questions concerning whether it can be used to infer evolutionary time from contemporary sequence differences. Received: 28 February 1995 / Accepted: 6 October 1997     

Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution.

The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule.     

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn^WT) and mouse (smFcRn^WT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn^WT bound human serum albumin with a K_D of ∼90 μm, whereas shFcRn^WT bound mouse serum albumin with a K_D of 0.8 μm. shFcRn^WT ignored mouse IgG1, and smFcRn^WT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.     

Natural variation in GA1 associates with floral morphology in Arabidopsis thaliana

? The genetic architecture of floral traits is evolutionarily important due to the fitness consequences of quantitative variation in floral morphology. Yet, little is known about the genes underlying these traits in natural populations. Using Arabidopsis thaliana, we examine molecular variation at GIBBERELLIC ACID REQUIRING 1 (GA1) and test for associations with floral morphology. ? We examined full-length sequence in 32 accessions and describe two haplotypes (comprising four nonsynonymous polymorphisms) in GA1 that segregate at intermediate frequencies. In 133 A. thaliana accessions, we test for genotype-phenotype associations and corroborate these findings in segregating progenies. ? The two common GA1 haplotypes were associated with the length of petals, stamens, and to a lesser extent style-stigma length. Associations were confirmed in a segregating progeny developed from 19 accessions. We find analogous results in recombinant inbred lines of the Bayreuth × Shahdara cross, which differ only at one of 4 SNPs, suggesting that this SNP may contribute to the observed association. ? Assuming GA1 causally affects floral organ size, it is interesting that adjacent petal and stamen whorls are most strongly affected. This pattern suggests that GA1 could contribute to the greater strength of petal-stamen correlations relative to other floral-length correlations observed in some Brassicaceous species.