structure ͉ cell growth T he insulin-like growth factor-binding protein (IGFBP) family comprises six soluble proteins (IGFBP1-6) of Ϸ250 residues that bind to IGFs with nanomolar affinities (1-4). Because of their sequence homology, IGFBPs are assumed to share a common overall fold and are expected to have closely related IGF-binding determinants. Each IGFBP can be divided into three distinct domains of approximately equal lengths: highly conserved cysteinerich N and C domains and a central linker domain unique to each IGFBP species. Both the N and C domains participate in the binding to IGFs, although the specific roles of each of these domains in IGF binding have not been decisively determined (1-13). The C-terminal domain may be responsible for preferences of IGFBPs for one species of IGF over the other (2, 3-7, 9-13); the C-terminal domain is also involved in regulation of the IGF-binding affinity through interaction with extracellular matrix components (1,2,14) and is most probably engaged in mediating IGF1-independent actions (1, 4, 14). The central linker domain is the least conserved region and has never been cited as part of the IGF-binding site for any IGFBP. This domain is the site of posttranslational modifications, specific proteolysis (4), and the acid-labile subunit (1) and extracellular matrix associations (1, 2, 14) known for IGFBPs. Proteolytic cleavage in this domain is believed to produce loweraffinity N-and C-terminal fragments that cannot compete with IGF receptors for IGFs, and, thus, the proteolysis is assumed to be the predominant mechanism for IGF release from IGFBPs (4, 9).However, recent studies indicate that the resulting N-and Cterminal fragments still can inhibit IGF activity and have functional properties that differ from those of the intact proteins (1, 3, 5, 9).The structure of the N-terminal domain of IGFBP-5, free (15) and complexed to IGF1 (16), was solved some time ago. More recently, low-resolution structures of the C-terminal domain of IGFBP6 (12) and its binding surface on IGF2 (3, 12) have been determined with NMR spectroscopy. There is, however, no x-ray structure of a ternary complex of the C-terminal domain of any IGFBPs bound to both the N-terminal domain and IGF, although the C-terminal fragment of IGFBP4 was crystallized recently (9), and also the x-ray structure of the isolated C-terminal fragment of IGFBP1 has been solved (17). We recently reported the x-ray structure of the ternary complex of the N-and C-terminal domains of IGFBP4 bound to IGF1 (10) and described ordered structures for the N-terminal domain of IGFBP4 and IGF1. The C domain was represented by disconnected patches of electron density and could not be interpreted. We describe here the long-sought, highresolution x-ray structure of a complex of the N-and C-terminal domains of IGFBP4 bound to IGF1. We also present the structure of the C-terminal domain of IGFBP1 bound to the N-terminal domain of IGFBP4 and IGF1 and the structure of the binary complex of the N-terminal domain of IGFBP4 (residues 1-92)...

Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, "side-to-side" and "straight-longitudinal," which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament.cytoskeleton | X-ray crystallography | fluorescence assay T he actin cytoskeleton is involved in many cellular processes, including cell motility, cell adhesion, endo/exocytosis, intracellular and membrane trafficking, and the maintenance of cell shape and polarity. These cellular functions require the dynamic remodeling of the actin cytoskeleton, which depends on the transition between monomeric actin (G-actin) and its filamentous state (F-actin) (1). The initiation of actin polymerization from free actin monomers requires nucleation factors that help to overcome the kinetic barrier for formation of actin dimers and trimers (2, 3). Three classes of actin-nucleation proteins have been identified until today: the Arp2/3 complex together with newly recognized nucleation-promoting factors such as WASH, WHAMM, and JMY (4-8), formins (9-12), and the third group of proteins that contain 17-27 amino acid long actin-binding motifs called the WH2 repeats-the name derived from the WASP (Wiskott-Aldrich syndrome protein) homology domain 2 (13-15). The third group consists of Spire (16), Cordon-bleu (17), and Leiomodin from muscle cells (18). The molecular mechanism of actin nucleation is well described for the Arp2/3 complex (19,20) and formins (21), whereas little molecular details are known about the nucleation by Spire.Spire was first identified as an actin-binding factor necessary for the correct establishment of polarity axes of the oocyte in Drosophila (22). It mediated actin-microtubule interactions and has important roles in membrane transport, although the upstream signaling pathways that regulate these functions have not been fully studied (23)(24)(25). Spire is a 1,020 amino acid long, multidomain protein (Fig. 1A); the most important domains, from the point of view of actin organization, are localized in the N-terminal part of the protein...

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs-even a single WH2 repeat-sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.cytoskeleton | nucleation T he first step in the assembly of actin filaments is nucleation (1, 2). Three major classes of nucleating proteins have been identified until today: the Arp2/3 complex together with newly identified nucleation-promoting factors such as WASH, WHAMM and JMY (3-7), formins (8, 9), and a third class which comprises the proteins commonly named tandem-monomer-binding nucleators (10). This last group of nucleators contains 17-27 amino acid long actin-binding motifs called the WH2 repeats-the name derived from the Wiskott-Aldrich syndrome protein homology domain 2 (11, 12). The group consists of Spire (13), , Leiomodin from muscle cells (15), JMY (6), and the recently discovered adenomatous polyposis coli (APC) protein (16). These proteins share the common ability, mediated by WH2 domains, to gather actin monomers into a nucleation complex, but the arrangement of nuclei might vary significantly among these nucleators. Spire contains four consecutive WH2 domains and is the most important representative member of the group. The molecular mechanism of actin nucleation is well described for the Arp2/3 complex (17) and formins (18), whereas several different mechanisms have been proposed for Spire (19)(20)(21)(22), Leiomodin (15), JMY (6), and the APC protein (16). The N-terminal domain of Spire (SpireNT, residues 1-520 in Drosophila melanogaster Spire; see Fig. S1) has the potential to form a string of four actin monomers through the interaction with four WH2 repeats (13,19,20). WH2 motifs are known to be intrinsically disordered, adopting an α-helical structure only upon binding to actin (23). Alignments of WH2 domains indicate that the most conserved regions are the LKK motif and an α-helix at the N terminus, which is shown to be the principal structural actin-binding element that binds to actin in its hydrophobic pocket between actin's subdomains 1 and 3 (20,24,25). The rest of the WH2, including the LKK motif, exten...

The structures of the N-terminal domains of two integrases of closely related but not identical asn tDNA-associated genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin biosynthesis and transport) and an Erwinia carotovora genomic island with yet unknown function, HAI7, have been resolved. Both integrases utilize a novel fourstranded ␤-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded ␤-sheets. Moreover, the ␤-sheets in Int HPI and Int HAI7 are longer than those in other integrases, and the structured helical N terminus is positioned perpendicularly to the large C-terminal helix. These differences strongly support the proposal that the integrases of the genomic islands make up a distinct evolutionary branch of the site-specific recombinases that utilize a unique DNA-binding mechanism.Genomic islands, together with temperate phages, integrative plasmids, transposons, and integrative conjugative elements, make up the group of mobile genetic elements that play an important role in bacterial quantum leap evolution and adaptation (1-3). Genomic islands that carry clustered genes encoding vital functions supply bacteria with additional capabilities to withstand and overcome host defenses and to improve fitness. Genomic islands are integrative elements that are not able to self-transfer and replicate. Typically, they are composed of functional and recombination modules. The recombination module consists of a tyrosine family integrase and two attachment sites involved in recombination. The integrase promotes attPϫ attB site-specific DNA recombination of the genomic islands into highly conserved tRNA-encoding genes (attB recombination targets) of the host genome and subsequent excision (4,5).It has been demonstrated that the N-terminal domain of phage integrases is responsible for specific recognition of the arm-type site sequence of the attachment sites, a step that is essential for activity of the catalytic C-terminal domain responsible for the strand exchange. Three-dimensional structures of the N-terminal domains of two prokaryotic integrases, namely bacteriophage integrase and Tn916 transposon integrase, have been determined. Although they do not share significant sequence homology, both adopt similar structures and recognize the arm-type DNA site by inserting their N-terminal domain into a major groove of DNA (6 -9). The N-terminal domain, consisting of a three-stranded antiparallel ␤-sheet, is proposed to be a new DNA-binding motif whose residue composition and position within the major DNA groove varied to alter specificity (6). Nevertheless, the genomic islands are evolutionarily divergent from phages and other mobile elements and represent a distinct mobile genetic element class. Moreover, island-encoded integrases are not closely related to phage integrases, as was expected previously (3, 10).Four closely related genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin b...

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