The structure of neurexin 1α reveals features promoting a role as synaptic organizer

α-neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The α-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how α-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1α extracellular domain (n1α) to 2.65 ?. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 ? structure of n1α carrying splice insert SS#3 in LNS4 reveals that SS#3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables α-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.     

Provenance of SET-Domain Histone Methyltransferases Through Duplication of a Simple Structural Unit

SET domains are protein lysine methyltransferases that methylate diverse proteins, such as, histones, Rubisco and cytochrome C. In particular, they play an important role in the dynamics of the eukaryotic chromatin and are present in several chromatin-associated proteins. Recently, structures of several SET domains have been solved, and they contain a conserved fold that is unrelated to previously characterized methyltransferases, which possess either Rossmann fold or SPOUT domains. Phylogenetic and phyletic-profile analysis of the SET domain suggests that it was an evolutionary “invention” of the eukaryotic lineage, with secondary lateral transfers to bacteria. We show that the conserved N- and C- terminal regions, which comprise the core barrel-like module of the SET domain, are symmetric repeats of a simple 3-stranded unit. Furthermore, the two symmetrically arranged repeats contribute to the binding sites for the two substrates of the SET domain. This suggests the SET domain arose from an ancestral dimer of this 3-stranded unit, with each unit probably functioning as generic-ligand binding structure. The divergence between the two repeat units appears to have arisen as a result of their interactions with the central module of the SET domain, which was inserted between the two repeats. One of the repeats appears to have acquired adaptations, which helped it to specialize in AdoMet binding, whereas the second repeat contributed to histone-interaction, and in orienting a crucial active site residue. The central module of the SET domain supplies a critical asparagine to the active site, and its structural features suggest that it may have also arisen from a further duplication of one of the repeats comprising the core barrel. However, it appears to have structurally diverged from the two canonical repeats due to the lack of an obligate dimerization partner. The spatial position of the two repeats in the ancestral dimer appears to have favored the formation of the structural knot typical of the SET domain. A comparable knot is seen in the SPOUT-domain methyltransferases, and this represents a case of convergent evolution of an active-site-associated configuration in two otherwise unrelated classes of methylases. Thus, the SET domain provides a model for the innovation of a complex enzymatic fold through the duplications of a structurally simple non-enzymatic unit.     

Provenance of SET-domain histone methyltransferases through duplication of a simple structural unit

SET domains are protein lysine methyltransferases that methylate diverse proteins, such as, histones, Rubisco and cytochrome C. In particular, they play an important role in the dynamics of the eukaryotic chromatin and are present in several chromatin-associated proteins. Recently, structures of several SET domains have been solved, and they contain a conserved fold that is unrelated to previously characterized methyltransferases, which possess either Rossmann fold or SPOUT domains. Phylogenetic and phyletic-profile analysis of the SET domain suggests that it was an evolutionary "invention" of the eukaryotic lineage, with secondary lateral transfers to bacteria. We show that the conserved N- and C- terminal regions, which comprise the core barrel-like module of the SET domain, are symmetric repeats of a simple 3-stranded unit. Furthermore, the two symmetrically arranged repeats contribute to the binding sites for the two substrates of the SET domain. This suggests the SET domain arose from an ancestral dimer of this 3-stranded unit, with each unit probably functioning as generic-ligand binding structure. The divergence between the two repeat units appears to have arisen as a result of their interactions with the central module of the SET domain, which was inserted between the two repeats. One of the repeats appears to have acquired adaptations, which helped it to specialize in AdoMet binding, whereas the second repeat contributed to histone-interaction, and in orienting a crucial active site residue. The central module of the SET domain supplies a critical asparagine to the active site, and its structural features suggest that it may have also arisen from a further duplication of one of the repeats comprising the core barrel. However, it appears to have structurally diverged from the two canonical repeats due to the lack of an obligate dimerization partner. The spatial position of the two repeats in the ancestral dimer appears to have favored the formation of the structural knot typical of the SET domain. A comparable knot is seen in the SPOUT-domain methyltransferases, and this represents a case of convergent evolution of an active-site-associated configuration in two otherwise unrelated classes of methylases. Thus, the SET domain provides a model for the innovation of a complex enzymatic fold through the duplications of a structurally simple non-enzymatic unit.     

Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Immunochemical analysis of the structure of the signature domains of thrombospondin-1 and thrombospondin-2 in low calcium concentrations

Thrombospondins (TSPs) undergo conformational changes upon removal of calcium. The eight C-type and five N-type calcium-binding repeats of TSP-2 form a circuitous wire that, in 2 mm calcium, interacts at its ends with more N-terminal epidermal growth factor (EGF)-like modules, EGF2 and EGF3, and the C-terminal lectin-like module. These components, along with the other EGF-like module(s), form the signature domain of TSPs. Characterization of conformation-sensitive epitopes of monoclonal antibodies to human TSP-2 and its TSP-1 homolog have given insights into the structure of the signature domain in the absence of calcium. The epitope for 4B6.13 anti-TSP-2 was localized to His-722 and Leu-703 in repeat 1C of the wire; recognition only occurred in constructs that included EGF3, the rest of the wire, and the lectin-like module and in the presence of calcium. The epitope for C6.7 anti-TSP-1 was localized to Glu-609 in the EGF2 module. The C6.7 epitope was preferentially recognized when EGF2 was expressed in the context of EGF1, EGF3, the wire, and the lectin-like module. Preferential recognition of the C6.7 epitope did not require calcium. Rotary shadowing electron microscopy of TSP-1 has shown elongation of the stalk and diminution of the C-terminal globule. We propose a model whereby at low calcium concentrations the lectin-like module drops away from EGF3 concomitant with changes in conformation of the wire and loss of the 4B6.13 epitope. A critical feature of the model is interaction of repeat 12N of the wire with EGF2 in both the presence and absence of calcium.     

A minimum folding unit in the ankyrin repeat protein p16(INK4)

The ankyrin repeat is an abundant, 33 residue sequence motif that forms a consecutive beta-hairpin-helix-loop-helix (beta(2)alpha(2)) fold. Most ankyrin repeat proteins consist of four or more complete repeats, which provide stabilizing interactions between adjacent modules. The cyclin-dependent kinase inhibitor and tumor suppressor p16(INK4) (p16) is one of the smallest ankyrin repeat proteins with a known structure. It consists of four complete repeats plus short N and C-terminal flanking regions that are unstructured in solution. On the basis of preliminary proteolysis studies and predictions using a computer algorithm for identifying autonomous folding units, we have identified a fragment consisting of the third and fourth ankyrin repeats of p16, called p16C, that can fold independently, without the rest of the protein. Far-UV circular dichroism studies showed that p16C has a significant level of alpha-helical secondary structure, and two proline substitutions that disrupt the alpha-helical secondary structure in wild-type p16 disrupt the secondary structure in p16C. The thermal denaturation of p16C is cooperative and reversible, with a midpoint of transition at 30. 5(+/-1) degrees C. From urea-induced denaturation studies, the free energy of unfolding for p16C was estimated to be 1.7(+/-0.3) kcal/mol at 20 degrees C. (1)H-(15)N 2D NMR studies suggest that the ankyrin repeats in p16C are likely to fold into a structure similar to that of full-length p16. In order to define the minimum autonomous folding unit in p16, we have further dissected p16C into two complementary peptides, each containing a single ankyrin repeat. These peptides are unstructured in solution. Thus, p16C is the smallest ankyrin repeat module that is known to fold independently and, in general, we believe that the two-ankyrin repeat fold could be the minimum structural unit for all ankyrin repeat proteins. We further discuss the significance of p16C in protein folding and engineering.     

Folding of spectrin's SH3 domain in the presence of spectrin repeats

The multifunctional protein spectrin contains several different structural motifs, such as spectrin repeats and a SH3 domain. Both triple-helix spectrin repeats and the SH3 domain are believed to form independent structural entities. In alpha-spectrins the SH3 domain is localized to repeat 9, where it is positioned between helix B and helix C in the repeat unit. The presence of SH3 in repeat 9 decreases the thermal stability considerably of this repeat unit while another insert in helix C does not seem to affect the stability. Addition of one or two adjacent repeat units increases the thermal stability from ca 25 degrees C to 41 and 48 degrees C, respectively. Despite the differences in thermal stability, the folding properties of peptides comprising the SH3 domain only or together with one or more repeats are more or less the same.     

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides

Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4. In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation. Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet.     

Structure of 1.71 lb gm/cm(3) bovine satellite DNA: evolutionary relationship to satellite I

The Eco RI fragments from the 2600 bp repeating unit of 1.711b gm/Cm(3) bovine satellite DNA were cloned in pBR322. The structure of the repeat unit was determined and compared to bovine satellite I DNA (rho CsCl = 1.715 gm/cm(3)). All of the DNA in the 1402 bp repeat of satellite I is represented in the sequence of the 2600 bp 1.711b gm/cm(3) repeat. The difference between the two repeats is due to a 1200 bp piece of DNA (INS) residing in the middle of the 1.711b gm/cm(3) repeat. The INS is AT-rich and has some repetitive components; it bears only limited similarity to the structure of eukaryotic transposable elements. We propose that the 1.711b gm/cm(3) satellite DNA arose via the amplification of a 1.715 gm/cm(3) satellite repeat altered by a 1200 bp insertion of DNA.     

Transfer origins in the conjugative Enterococcus faecalis plasmids pAD1 and pAM373: identification of the pAD1 nic site,a specific relaxase and a possible TraG-like protein

The Enterococcus faecalis conjugative plasmids pAD1 and pAM373 encode a mating response to the peptide sex pheromones cAD1 and cAM373 respectively. Sequence determination of both plasmids has recently been completed with strong similarity evident over many of the structural genes related to conjugation. pAD1 has two origins of transfer, with oriT1 being located within the repA determinant, whereas the more efficiently utilized oriT2 is located between orf53 and orf57, two genes found in the present study to be essential for conjugation. We have found a similarly located oriT to be present in pAM373. oriT2 corresponds to about 285 bp based on its ability to facilitate mobilization by pAD1 when ligated to the shuttle vector pAM401; however, it was not mobilized by pAM373. In contrast, a similarly ligated fragment containing the oriT of pAM373 did not facilitate mobilization by pAD1 but was efficiently mobilized by pAM373. The oriT sites of the two plasmids each contained a homologous large inverted repeat (spanning about 140 bp) adjacent to a series of non-homologous short (6 bp) direct repeats. A hybrid construction containing the inverted repeat of pAM373 and direct repeats of pAD1 was mobilized efficiently by pAD1 but not by pAM373, indicating a significantly greater degree of specificity is associated with the direct repeats. Mutational (deletion) analyses of the pAD1 oriT2 inverted repeat structure suggested its importance in facilitating transfer or perhaps ligation of the ends of the newly transferred DNA strand. Analyses showed that Orf57 (to be called TraX) is the relaxase, which was found to induce a specific nick in the large inverted repeat inside oriT; the protein also facilitated site-specific recombination between two oriT2 sites. Orf53 (to be called TraW) exhibits certain structural similarities to TraG-like proteins, although there is little overall homology.     

Domain Swapping Reveals That Low Density Lipoprotein (LDL) Type A Repeat Order Affects Ligand Binding to the LDL Receptor

The low density lipoprotein receptor (LDLR) plays a key role in plasma cholesterol homeostasis by binding and internalizing lipoprotein ligands. Studies have revealed that one or more of the seven LDL type A repeats (LA1–LA7) in the receptor are responsible for apolipoprotein binding. In the present study, protein engineering was performed to swap or replace key LA repeats in a recombinant soluble LDLR (sLDLR). Although wild type sLDLR showed strong ligand binding activity, an sLDLR variant in which LA repeat 5 was replaced by a second copy of LA repeat 2 showed low binding activity. Likewise, a variant wherein LA repeats 2 and 5 were swapped displayed low binding activity. At the same time, substitution of LA repeat 2 with a second a copy of repeat 5 resulted in a receptor with ligand binding activity similar to wild type LDLR. When binding assays were conducted with human low density lipoprotein as ligand, LA repeat order was a less important determinant of binding activity. Taken together, the data indicate that the sequential order of LA repeats plays a key role in ligand binding properties of LDLR.The low density lipoprotein receptor (LDLR)³ plays an important role in plasma cholesterol homeostasis (1). A fundamental function of LDLR is transport of cholesterol-rich lipoproteins into cells via receptor-mediated endocytosis (2). Human LDLR is 839 amino acids in length and is comprised of five distinct modules that arose from gene duplication. At the N terminus of LDLR, there exists a series of seven imperfect, disulfide bond-rich, LDL type A (LA) repeats, each ∼40 amino acids in length. Calcium binding induces LA repeats to fold into a ligand binding-competent conformation (3). Adjacent to the ligand binding module is a ∼400-residue module that bears homology to epidermal growth factor (EGF) precursor. This module consists of two disulfide bond-rich EGF-like repeats (A and B) and a YWTD β-propeller motif followed by a third EGF-like repeat C (4). The third module of LDLR is distinguished by an abundance of O-linked sugars, whereas the fourth module is comprised of a single membrane-spanning sequence. Finally, a short intracellular C-terminal cytoplasmic domain, required for receptor internalization, is present (5).LDLR binds two apolipoprotein ligands, apolipoprotein (apo) E and apoB (6). Although these proteins do not share structural similarity, sequence elements rich in positively charged amino acid side chains are present in each that are required for binding. Deletion studies have demonstrated that specific LA repeats are required for apolipoprotein binding to LDLR (7, 8).Recently, another LDLR ligand, termed proprotein convertase subtilisin-like kexin type 9 (PCSK9), has emerged (9): PCSK9 serves to regulate cholesterol homeostasis by modulating LDLR processing. Unlike lipoprotein ligands, PCSK9 binds EGF repeat A and, apparently, is not released from the receptor at endosomal pH.LA1–LA7 are ∼40–50% identical in primary sequence. Each repeat contains a Ca²⁺ binding site and three disulfide bonds. The importance of these structural features for ligand binding is widely recognized. For example, it is known that LA5 is essential for optimal binding of apoB- and apoE-containing ligands (7, 8). On the other hand, deletion of LA2 had no effect on binding of apoE-containing lipoproteins. X-ray crystal structure information is available for isolated LA5 at pH 5.0 (10) as well as the entire ectodomain of LDLR (residues 1–699) at endosomal pH (11). Based on this structural information and complementary data on apolipoprotein ligands, it has been postulated that electrostatic interactions modulate LDLR conformation and ligand binding. Given this, it remains unclear whether the precise order of LA repeats within the ligand binding module may impact ligand binding.In the present study, protein engineering of a soluble LDLR (sLDLR) was performed to swap or replace specific LA repeats within the ligand binding module of sLDLR. Ligand binding to wild type (WT) and engineered sLDLR was then determined. The results show that LA repeat 5 must not only be present, it must exist in the correct context with respect to other LA repeats within the ligand binding module.     

Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).     

Constant and variable parts of the 155-bp centromeric repeat in Camptochironomus

Centromeres in dipteran insects belonging to the subgenus Camptochironomus (C. tentans and C. pallidivittatus) contain tandem repeats of a 155-bp repeat. A special structural feature of the 155-bp unit is a region with two palindromes connected by a short piece of DNA with only AT base pairs, which has at least a superficial similarity to a functionally important part of the Saccharomyces cerevisiae centromere. As a parameter for functional importance we have measured frequencies of mutations along the unit in samples of repeats from the two species. We find that it consists of about equal parts of highly conserved and considerably less-conserved DNA. The palindromes are localized in the conserved part of the repeat. Correspondence to: J.-E. Edström     

The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport

Importin-beta is a nuclear transport factor which mediates the nuclear import of various nuclear proteins. The N-terminal 1-449 residue fragment of mouse importin-beta (impbeta449) possesses the ability to bidirectionally translocate through the nuclear pore complex (NPC), and to bind RanGTP. The structure of the uncomplexed form of impbeta449 has been solved at a 2.6 A resolution by X-ray crystallography. It consists of ten copies of the tandemly arrayed HEAT repeat and exhibits conformational flexibility which is involved in protein-protein interaction for nuclear transport. The overall conformation of the HEAT repeats shows that a twisted motion produces a significantly varied superhelical architecture from the previously reported structure of RanGTP-bound importin-beta. These conformational changes appear to be the sum of small conformational changes throughout the polypeptide. Such a flexibility, which resides in the stacked HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that impbeta449 has a structural similarity with another nuclear migrating protein, namely beta-catenin, which is composed of another type of helix-repeated structure of ARM repeat. Interestingly, the essential regions for NPC translocation for both importin-beta and beta-catenin are spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage.     

Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like beta-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.     

Domain structure and interactions of recombinant urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) is a mosaic glycoprotein composed of an epidermal growth factor-like (EGF), a kringle and a serine protease (SP) module. It exists in single and two-chain forms designated HMW pro-uPA and HMW uPA, respectively. A low molecular weight form, LMW uPA, lacks the EGF and kringle modules and is composed of the SP module alone. Recombinant-expressed proteins representing both HMW forms exhibit four reversible unfolding transitions that are resolved by deconvolution of melting curves obtained by differential scanning calorimetry at pH 4.5; no differences in the melting properties of the single and two-chain forms were found. The proteolytic fragment Ser1-Lys135 (EGF-kringle) exhibits two transitions, while the isolated EGF and kringle modules each exhibit a single two-state transition. Thus, both of these modules retain an independently folded compact structure when isolated. The isolated SP module (LMW uPA) exhibits two closely spaced transitions at low pH indicating the melting of two domains of similar stability. Fluorescence-detected melting curves of LMW uPA reveal increasing cooperativity with increasing pH, suggesting an increase in the interaction between the two SP domains. Treatment of both HMW and LMW uPA with the tripeptide inhibitor Glu-Gly-Arg-chloromethylketone dramatically increased the stability of both domains of the SP module which now melt together in a single two-state transition, even at low pH, with no effect on the EGF and kringle modules. From these data one concludes that UK consists of four independently folded domains. Two are formed by the EGF and kringle modules which do not interact with each other or with the SP module. The SP module contains two domains that are independent at low pH but exhibit a tendency to merge into a single cooperative unit at neutral pH or after treatment with the tripeptide inhibitor.     

A helical string of alternately connected three-helix bundles for the cell wall-associated adhesion protein Ebh from Staphylococcus aureus

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.