Functional mapping of an oligomeric autotransporter adhesin of Aggregatibacter actinomycetemcomitans

Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.     

The extended signal peptide of the trimeric autotransporter EmaA of Aggregatibacter actinomycetemcomitans modulates secretion

The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in Escherichia coli and A. actinomycetemcomitans using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in E. coli. A. actinomycetemcomitans strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.     

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.     

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.     

Investigation of the Three-Dimensional Architecture of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans by Electron Tomography

The periodontal pathogen Aggregatibacter actinomycetemcomitans displays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140° and 170° at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of ∼5 nm and a cylindrical domain (∼4.4 nm by 5.8 nm) separated by a linker region with a diameter of ∼3 nm, followed by a cylindrical domain (∼4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein of A. actinomycetemcomitans.Bacterial adhesion to host receptors, a crucial step for colonization and infection, is mediated by fimbrial and nonfimbrial adhesins. These adhesins are proteinaceous appendages displayed on the surface of bacteria and contain the receptor binding domains. Aggregatibacter actinomycetemcomitans, a gram-negative, nonmotile bacterium is found associated with periodontal diseases and other extraoral infections (12, 23, 32, 40). When isolated from the oral cavity, the bacterium exists as a fimbriated form and switches to an afimbriated form upon planktonic subculturing (5, 14). A. actinomycetemcomitans fimbriae mediate the nonspecific adherence of the bacterium to abiotic and organic surfaces and decorate the bacterial surface with long fibrils of 5 to 7 nm in diameter (14, 15). In addition to fimbriae, nonfimbrial adhesins, which mediate the specific binding to host cells and tissues, have been identified in this bacterium (1, 6, 19, 27, 29). Among these nonfimbrial adhesins, only the extracellular matrix protein adhesin A, EmaA, has been visualized forming structures on the bacterial surface by transmission electron microscopy (29).EmaA is an outer membrane collagen adhesin unique to A. actinomycetemcomitans; however, orthologous proteins exist in other bacterial genera (13, 18, 21, 26, 33, 38). The protein is encoded by a 6-kb gene present in all A. actinomycetemcomitans strains investigated (36). Genetic heterogeneity within the gene exists between different strains, which are based on the serotype of the organism. Based on this heterogeneity, two molecular forms of the protein have been identified: a full-length and an intermediate form. The prototypic or full-length protein exists as a 202-kDa protein and shares 75% amino acid homology with the intermediate form. The intermediate protein form (173 kDa) contains an in-frame 279-amino-acid deletion but maintains collagen binding activity and surface appendages similar to the prototypic form (36).EmaA is associated with the binding of A. actinomycetemcomitans to both isolated acid-soluble collagen and collagen found in tissues (19, 29, 35, 39). The specificity of EmaA for collagen was demonstrated using a rabbit cardiac valve tissue model (35). Valves with an intact endothelium bound equal amounts of the wild type or emaA isogenic mutants. Removal of the endothelium by trypsin treatment, thereby exposing the underlying collagen, did not affect the level of binding of the mutant. However, the number of wild-type bacteria bound to the exposed collagen was five times the number of mutant bacteria. This represents a 10-fold increase with respect to the number of bacteria bound to the endothelium. The role of EmaA as a virulence determinant in A. actinomycetemcomitans infection was demonstrated in a rabbit endocarditis infection model, in which the wild-type bacterium outcompeted the binding of the mutant 10-fold (35).Sequence analysis indicates that EmaA belongs to the Oca (oligomeric coiled-coil adhesin) family of autotransporter adhesins (19). Multimers of EmaA oligomerize to form appendages on the bacterial surface and are visible as long rods or antenna-like structures capped by an ellipsoidal domain (29). A strong correlation exists between the translocated region of the protein (head and stalk domains) and the structural features. The head domain, consisting of amino acids 70 to 386, forms the ellipsoidal ending of the appendage, which is essential for collagen binding, while amino acids 387 to 1900 form the stalk domain (39).Contained within the translocation domain of EmaA are three “neck” sequences, which are conserved in the Oca family protein members (21, 29, 33). These sequences are considered to stabilize the oligomer and transition between β-rolls and coiled-coil regions of the molecule (21, 26). In the EmaA sequence, two “neck” sequences are found within the first 628 amino acids of the protein sequence (19, 29). The third sequence is located in the stalk domain adjacent to the carboxy-terminal membrane anchor domain, which comprises amino acids 1901 to 1965 (19, 29). The membrane anchor domains of three or four monomers are proposed to form β-barrels that are required for pore formation and protein translocation (18, 29, 37).The translocated domain of EmaA has been subjected to a two-dimensional (2D) study by transmission electron microscopy, and the overall dimensions of the EmaA appendages have been determined by the analysis of a large number of micrographs (29). The ellipsoidal ending shows diameters of 2.8 by 4.6 nm, and the stalk domain, which is at least 150 nm long, has a diameter of 4.1 nm. Several conformations of the stalk domain were present in the micrographs: either straight or containing a bend at 29.2 nm from the distal end. This bend position was correlated with amino acids localized between the first two neck sequences (29).In this study, electron tomography was used to characterize the 3D structure of the EmaA appendages of A. actinomycetemcomitans in situ. The functional domain of EmaA was found to be composed of three distinct subdomains followed by a long stalk domain. Distinct regions of the molecule were identified that provide flexibility for the molecule and allow for the deformation or bending of the adhesin. A correlation between these flexible regions and specific amino acids in the sequence was ascertained.     

Structure of the tetraspanin main extracellular domain. A partially conserved fold with a structurally variable domain insertion

The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold.     

Myosin V attachment to cargo requires the tight association of two functional subdomains

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle-specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.     

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.     

NMR structure of an F-actin-binding "headpiece" motif from villin

A growing family of F-actin-bundling proteins harbors a modular F-actin-binding headpiece domain at the C terminus. Headpiece provides one of the two F-actin-binding sites essential for filament bundling. Here, we report the first structure of a functional headpiece domain. The NMR structure of chicken villin headpiece (HP67) reveals two subdomains that share a tightly packed hydrophobic core. The N-terminal subdomain contains bends, turns, and a four-residue alpha-helix as well as a buried histidine residue that imparts a pH-dependent folding. The C-terminal subdomain is composed of three alpha-helices and its folding is pH-independent. Two residues previously implicated in F-actin-binding form a buried salt-bridge between the N and C-terminal subdomains. The rest of the identified actin-binding residues are solvent-exposed and map onto a unique F-actin-binding surface.     

Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotide-free and nucleotide-bound conformations

The ABC-ATPase GlcV energizes a binding protein-dependent ABC transporter that mediates glucose uptake in Sulfolobus solfataricus. Here, we report high-resolution crystal structures of GlcV in different states along its catalytic cycle: distinct monomeric nucleotide-free states and monomeric complexes with ADP-Mg(2+) as a product-bound state, and with AMPPNP-Mg(2+) as an ATP-like bound state. The structure of GlcV consists of a typical ABC-ATPase domain, comprising two subdomains, connected by a linker region to a C-terminal domain of unknown function. Comparisons of the nucleotide-free and nucleotide-bound structures of GlcV reveal re-orientations of the ABCalpha subdomain and the C-terminal domain relative to the ABCalpha/beta subdomain, and switch-like rearrangements in the P-loop and Q-loop regions. Additionally, large conformational differences are observed between the GlcV structures and those of other ABC-ATPases, further emphasizing the inherent flexibility of these proteins. Notably, a comparison of the monomeric AMPPNP-Mg(2+)-bound GlcV structure with that of the dimeric ATP-Na(+)-bound LolD-E171Q mutant reveals a +/-20 degrees rigid body re-orientation of the ABCalpha subdomain relative to the ABCalpha/beta subdomain, accompanied by a local conformational difference in the Q-loop. We propose that these differences represent conformational changes that may have a role in the mechanism of energy-transduction and/or allosteric control of the ABC-ATPase activity in bacterial importers.     

Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.     

Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity

Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis. Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI. However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity. In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria. Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI. The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains. However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed. The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX.     

The role of the LH subdomain in the function of the Cip/Kip cyclin-dependent kinase regulators

The Cip/Kip protein family, which includes p27, p21, and p57, modulates the activity of cyclin-dependent kinases (Cdks). A domain within these proteins, termed the kinase inhibitory domain (KID), is necessary and sufficient for Cdk inhibition. The KID consists of a cyclin-binding subdomain (termed D1) and a Cdk-binding subdomain (termed D2) joined by a 22-residue linker subdomain (termed LH). Before binding the Cdks, D1 and D2 are largely unstructured and the LH subdomain exhibits nascent helical characteristics. Curiously, although the sequence of the linker subdomain is not highly conserved within the family, its nascent helical structure is conserved. In this study, we explored the role of this structural conservation in interactions with cyclin-dependent kinase 2 (Cdk2) and cyclin A. We constructed chimeric p27-KID molecules in which the p27 LH subdomain was replaced with the corresponding segments of either p21 or p57. The chimeric molecules bind and inhibit Cdk2 in a manner similar to wild-type p27-KID. However, the extent of enthalpy/entropy compensation associated with these interactions was dramatically different, indicating different extents of LH subdomain folding upon binding. Our results indicate that the different LH subdomains, despite their sequence and thermodynamic differences, play similar roles in binding and inhibiting Cdk2/cyclin A.     

A point mutation in the cargo-binding domain of myosin V affects its interaction with multiple cargoes

Class V myosins move diverse intracellular cargoes, which attach via interaction of cargo-specific proteins to the myosin V globular tail. The globular tail of the yeast myosin V, Myo2p, contains two structural and functional subdomains. Subdomain I binds to the vacuole-specific protein, Vac17p, while subdomain II likely binds to an as yet unidentified secretory vesicle-specific protein. All functions of Myo2p require the tight association of subdomains I and II, which suggests that binding of a cargo to one subdomain may inhibit cargo-binding to a second subdomain. Thus, two types of mutations are predicted to specifically affect a subset of Myo2p cargoes: first are mutations within a cargo-specific binding region; second are mutations that mimic the inhibited conformation of one of the subdomains. Here we analyze a point mutation in subdomain I, myo2-2(G1248D), which is likely to be this latter type of mutation. myo2-2 has no effect on secretory vesicle movement. The secretory vesicle binding site is in subdomain II. However, myo2-2 is impaired in several Myo2p-related functions. While subdomains I and II of myo2-2p tightly associate, there are measurable differences in the conformation of its globular tail. Based solely on the ability to restore vacuole inheritance, a set of intragenic suppressors of myo2-2 were identified. All suppressor mutations reside in subdomain I. Moreover, subdomain I and II interactions occurred in all suppressors, demonstrating the importance of subdomain I and II association for Myo2p function. Furthermore, 3 of the 10 suppressors globally restored all tested defects in myo2-2. This large proportion of global suppressors strongly suggests that myo2-2(G1248) causes a conformational change in subdomain I that simultaneously affects multiple cargoes.     

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

The structural organization of the hamster multifunctional protein CAD. Controlled proteolysis, domains, and linkers.

CAD is a multidomain protein that catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The 243-kDa polypeptide consists of four functional domains; glutamine amidotransferase (GLNase), carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase). Controlled proteolysis of hamster CAD was found to cleave the molecule into 18 fragments which successively accumulate and disappear during the course of digestion. Each fragment was isolated and partially sequenced to determine its location in the polypeptide chain. Proteolysis was found to usually occur at the junctions between the domains and sub-domains identified by sequence homology. All proteases of low to moderate specificity cleaved the molecule in a similar fashion. The rate of proteolysis widely varied and the interdomain regions were not always accessible to proteases. Each of the major functional domains is postulated to consist of subdomains. The duplicated halves of the CPSase domain (116 kDa) have a homologous structure consisting of 11-, 25-26-, and 21-22-kDa subdomains. Prolonged digestion cleaved the DHOase domain (36.6 kDa) into two stable species suggesting that this region is comprised of 11.5- and 15.0-kDa subdomains. Similarly, proteolysis of the 21-kDa catalytic subdomain of the GLNase domain (40 kDa) indicated a bilobal structure consisting of 12.3- and 8.5-kDa chain segments. The connecting region between the two ATCase subdomains (16.4 and 18 kDa) was not cleaved. Copurification of many of the domains showed that they remain associated by noncovalent interactions even after the connecting segments have been cleaved. The chain segments, the linkers, which connect the domains and subdomains were conserved in length but not in sequence, were predicted to be relatively hydrophilic and flexible but did not show a tendency to assume a particular secondary structure. These studies provide a more detailed map of the structural organization of the CAD polypeptide.