NORMAL GENOMIC

• Cramer JF, Nordberg PA, Hajdu J, Lejon S
• Crystal structure of a bacterial albumin-binding domain at 1.4 Aresolution.
• FEBS Lett. 2007; 581: 3178-82
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• The albumin-binding domain, or GA module, of the peptostreptococcalalbumin-binding protein expressed in pathogenic strains of Finegoldiamagna is believed to be responsible for the virulence and increased growthrate of these strains. Here we present the 1.4A crystal structure of thisdomain, and compare it with the crystal structure of the GA-albumincomplex. An analysis of protein-protein interactions in the two crystals,and the presence of multimeric GA species in solution, indicate the GAmodule is "sticky", and is capable of forming contacts with a range ofprotein surfaces. This might lead to interactions with different hostproteins.

• He Y, Chen Y, Rozak DA, Bryan PN, Orban J
• An artificially evolved albumin binding module facilitates chemical shiftepitope mapping of GA domain interactions with phylogenetically diversealbumins.
• Protein Sci. 2007; 16: 1490-4
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• Protein G-related albumin-binding (GA) modules occur on the surface ofnumerous Gram-positive bacterial pathogens and their presence may promotebacterial growth and virulence in mammalian hosts. We recently used phagedisplay selection to evolve a GA domain, PSD-1 (phage selected domain-1),which tightly bound phylogenetically diverse albumins. With respect toPSD-1's broad albumin binding specificity, it remained unclear how theevolved binding epitope compared to those of naturally occurring GAdomains and whether PSD-1's binding mode was the same for differentalbumins. We investigate these questions here using chemical shiftperturbation measurements of PSD-1 with rabbit serum albumin (RSA) andhuman serum albumin (HSA) and put the results in the context of previouswork on structure and dynamics of GA domains. Combined, these data provideinsights into the requirements for broad binding specificity in GA-albumininteractions. Moreover, we note that using the phage-optimized PSD-1protein significantly diminishes the effects of exchange broadening at thebinding interface between GA modules and albumin, presumably throughstabilization of a ligand-bound conformation. The employment ofartificially evolved domains may be generally useful in NMR structuralstudies of other protein-protein complexes.

• Lejon S, Frick IM, Bjorck L, Wikstrom M, Svensson S
• Crystal structure and biological implications of a bacterial albuminbinding module in complex with human serum albumin.
• J Biol Chem. 2004; 279: 42924-8
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• Many bactericide species express surface proteins that interact with humanserum albumin (HSA). Protein PAB from the anaerobic bacterium Finegoldiamagna (formerly Peptostreptococcus magnus) represents one of theseproteins. Protein PAB contains a domain of 53 amino acid residues known asthe GA module. GA homologs are also found in protein G of group C and Gstreptococci. Here we report the crystal structure of HSA in complex withthe GA module of protein PAB. The model of the complex was refined to aresolution of 2.7 A and reveals a novel binding epitope located in domainII of the albumin molecule. The GA module is composed of a left-handedthree-helix bundle, and residues from the second helix and the loopssurrounding it were found to be involved in HSA binding. Furthermore, thepresence of HSA-bound fatty acids seems to influence HSA-GA complexformation. F. magna has a much more restricted host specificity comparedwith C and G streptococci, which is also reflected in the binding ofdifferent animal albumins by proteins PAB and G. The structure of theHSA-GA complex offers a molecular explanation to this unusually clearexample of bacterial adaptation.

• Johansson MU et al.
• Differences in backbone dynamics of two homologous bacterialalbumin-binding modules: implications for binding specificity andbacterial adaptation.
• J Mol Biol. 2002; 316: 1083-99
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• Proteins G and PAB are bacterial albumin-binding proteins expressed at thesurface of group C and G streptococci and Peptostreptococcus magnus,respectively. Repeated albumin-binding domains, known as GA modules, arefound in both proteins. The third GA module of protein G from the group Gstreptococcal strain G148 (G148-GA3) and the second GA module of proteinPAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity andboth fold to form three-helix bundle structures that are very stableagainst thermal denaturation. ALB8-GA binds human serum albumin withhigher affinity than G148-GA3, but G148-GA3 shows substantially broaderalbumin-binding specificity than ALB8-GA. The (15)N nuclear magneticresonance spin relaxation measurements reported here, show that the two GAmodules exhibit mobility on the picosecond-nanosecond time scale indirectly corresponding regions (loops and termini). Most residues inG148-GA3 were seen to be involved in conformational exchange processes onthe microsecond-millisecond time scale, whereas for ALB8-GA such motionswere only identified for the beginning of helix 2 and its preceding loop.Furthermore, and more importantly, hydrogen-deuterium exchange andsaturation transfer experiments reveal large differences between the twoGA modules with respect to motions on the second-hour time scale. The highdegree of similarity between the two GA modules with respect to sequence,structure and stability, and the observed differences in dynamics, bindingaffinity and binding specificity to different albumins, suggest a distinctcorrelation between dynamics, binding affinity and binding specificity.Finally, it is noteworthy in this context that the module G148-GA3, whichhas broad albumin-binding specificity, is expressed by group C and Gstreptococci known to infect all mammalian species, whereas P.magnus withthe ALB8-GA module has been isolated only from humans.

• Johansson MU et al.
• Structure, specificity, and mode of interaction for bacterialalbumin-binding modules.
• J Biol Chem. 2002; 277: 8114-20
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• We have determined the solution structure of an albumin binding domain ofprotein G, a surface protein of group C and G streptococci. We find thatit folds into a left handed three-helix bundle similar to the albuminbinding domain of protein PAB from Peptostreptococcus magnus. The twodomains share 59% sequence identity, are thermally very stable, and bindto the same site on human serum albumin. The albumin binding site, thefirst determined for this structural motif known as the GA module,comprises residues spanning the first loop to the beginning of the thirdhelix and includes the most conserved region of GA modules. The two GAmodules have different affinities for albumin from different species, andtheir albumin binding patterns correspond directly to the host specificityof C/G streptococci and P. magnus, respectively. These studies of theevolution, structure, and binding properties of the GA module emphasizethe power of bacterial adaptation and underline ecological and medicalproblems connected with the use of antibiotics.

• de Chateau M, Holst E, Bjorck L
• Protein PAB, an albumin-binding bacterial surface protein promoting growthand virulence.
• J Biol Chem. 1996; 271: 26609-15
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• The anaerobic bacterium Peptostreptococcus magnus is a human commensal andpathogen. Previous work has shown that strains of P. magnus isolated frompatients with gynecological disease (vaginosis) frequently express animmunoglobulin (Ig) light chain-binding protein called protein L. Here wereport that strains isolated from localized suppurative infections bindhuman serum albumin (HSA), whereas commensal isolates bind neither Ig norHSA. The HSA-binding protein PAB was extracted from the bacterial surfaceor isolated from the culture supernatant of the P. magnus strain ALB8.Protein PAB was shown to have two homologous HSA-binding domains, GA anduGA. GA is absent in the sequence of a related protein from another P.magnus strain and shows a high degree of homology to the HSA-bindingdomains of streptococcal protein G. Therefore GA is believed to haverecently been shuffled as a module from genes of other bacterial speciesinto the protein PAB gene. This GA module was shown to exhibit a muchhigher affinity for HSA than uGA and was also found to be present in allof the isolates tested from localized suppurative infections, indicating arole in virulence. Moreover, when peptostreptococci or streptococciexpressing the GA module were grown in the presence of HSA, the growthrate was substantially increased. Thus, the HSA binding activity of the GAmodule adds selective advantages to the bacteria, which increases theirvirulence in the case of P. magnus strains.

• Johansson MU, de Chateau M, Bjorck L, Forsen S, Drakenberg T, Wikstrom M
• The GA module, a mobile albumin-binding bacterial domain, adopts athree-helix-bundle structure.
• FEBS Lett. 1995; 374: 257-61
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• We present the first study of the secondary structure and global fold ofan albumin-binding domain. Our data show that the GA module from proteinPAB, an albumin-binding protein from the anaerobic bacterial speciesPeptostreptococcus magnus, is composed of a left-handed three-helixbundle. The helical regions were identified by sequential and medium rangeNOEs, values of NH-C alpha H coupling constants, chemical shift indices,and the presence of slowly exchanging amide protons, as determined by NMRspectroscopy. In addition, circular dichroism studies show that the moduleis remarkably stable with respect to both pH and temperature.

• Wikstrom M, Sjobring U, Drakenberg T, Forsen S, Bjorck L
• Mapping of the immunoglobulin light chain-binding site of protein L.
• J Mol Biol. 1995; 250: 128-33
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• Protein L is a cell surface protein expressed by some strains of theanaerobic bacterial species Peptostreptococcus magnus. The molecule bindsspecifically and with high affinity to immunoglobulins (Ig) of a widerange of animal species. The Ig-binding activity is mediated through fivehighly homologous domains, each 72 to 76 amino acid residues long, whichinteract with framework regions in the variable domain of Ig light chains.The interaction does not interfere with the antigen binding capacity ofthe antibody. The fold of the Ig light chain-binding domains of Protein Lis comprised of an alpha-helix packed against a four stranded beta-sheetand is similar to the fold of the IgG heavy chain-binding domains ofstreptococcal protein G, despite the fact that the two proteins show nosignificant sequence homology. In the present work, heteronuclear NMRspectroscopy has been utilized to define the interaction between theN-terminal Ig-binding domain of Protein L and the variable domain of ahuman Ig kappa light chain. The Ig-binding region of the Protein L domaininvolves most of the residues in the second beta-strand, the C-terminalresidues of the alpha-helix and the loop connecting the alpha-helix withthe third beta-strand. The Ig light chain-binding surface of Protein Lthus resembles the surface of Protein G which binds to the C gamma 1domain of IgG, but is different from the portion of Protein G involved inthe contact with the C gamma 2-C gamma 3 interface region. The datasuggest that the global fold shared by the Ig-binding domains of ProteinsL and G provide bacteria with a flexible template for the evolution ofsurface structures capable of interacting with different conserved partsof Ig molecules of the infected host.

• de Chateau M, Bjorck L
• Protein PAB, a mosaic albumin-binding bacterial protein representing thefirst contemporary example of module shuffling.
• J Biol Chem. 1994; 269: 12147-51
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• Some strains of the anaerobic human commensal and pathogenPeptostreptococcus magnus bind human serum albumin (HSA), whereas otherstrains of this species express protein L, an immunoglobulin lightchain-binding surface protein. A novel HSA-binding protein called proteinPAB was purified in one step from the culture supernatant of anHSA-binding strain of P. magnus by affinity chromatography onHSA-Sepharose. The apparent size of the molecular was 47 kDa onSDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis ofprotein PAB demonstrated that the 4 NH2-terminal residues were identicalto the corresponding sequence in protein L. In a polymerase chainreaction, oligonucleotides based on extragenic 5'- and 3'-end sequences ofthe protein L gene generated a product of the expected size: 1.3 kilobasepairs. A recombinant protein with retained albumin binding capacity wasexpressed in Escherichia coli, and the nucleotide sequence of the proteinPAB gene was determined. The structural gene is 1161 nucleotides long,corresponding to a preprotein of 387 amino acids and a molecular mass of43,043 Da. Unlike most other Gram-positive bacterial surface proteinsdescribed, protein PAB contains no internal homologies. However,substantial homologies were found to both proteins L and G (the IgG- andHSA-binding surface protein of group C and G streptococci). The derivedamino acid sequence of the 135-base pair-long region homologous to proteinG corresponds to the HSA-binding domain of that protein, and in proteinPAB, this region is inserted between sequences showing extensive homologyto COOH-terminal regions of peptostreptococcal protein L. This mosaicorganization of protein PAB demonstrates that the molecule is a product ofintergenic interspecies recombination of a functional domain into a commonframework for peptostreptococcal surface proteins. Such an interspeciesexchange of a functional protein module has previously not been describedin prokaryotic cells.

• Wikstrom M, Drakenberg T, Forsen S, Sjobring U, Bjorck L
• Three-dimensional solution structure of an immunoglobulin lightchain-binding domain of protein L. Comparison with the IgG-binding domainsof protein G.
• Biochemistry. 1994; 33: 14011-7
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• Protein L is a multidomain protein expressed at the surface of somestrains of the anaerobic bacterial species Peptostreptococcus magnus. Ithas affinity for immunoglobulin (Ig) through interaction with frameworkstructures in the variable Ig light chain domain. The Ig-binding activityis located to five homologous repeats called B1-B5 in the N-terminal partof the protein. We have determined the three-dimensional solutionstructure of the 76 amino acid residue long B1 domain using NMRspectroscopy and distance geometry-restrained simulated annealing. Thedomain is composed of a 15 amino acid residue long disordered N-terminusfollowed by a folded portion comprising an alpha-helix packed against afour-stranded beta-sheet. These secondary structural elements are welldetermined with a backbone atomic root mean square deviation from theirmean of 0.54 A. The B domains of protein L show very limited sequencehomology to the domains of streptococcal protein G interacting with theheavy chains of IgG. However, despite this fact, and their differentbinding properties, the fold of the B1 domain was found to be similar tothe fold of the IgG-binding protein G domains [Wikstrom, M., Sjobring, U.,Kastern, W., Bjorck, L., Drakenberg, T., & Forsen, S. (1993) Biochemistry32, 3381-3386]. In the present study, the solution structure of the B1domain enabled a more detailed comparison which can explain the differentIg-binding specificities of these two bacterial surface proteins. Amongthe differences observed, the alpha-helix orientation is the moststriking.(ABSTRACT TRUNCATED AT 250 WORDS)