The gelsolin:calponin complex nucleates actin filaments with distinct morphologies

Gelsolin and calponin are cytoskeletal and signalling proteins that form a tight 1:1 complex (GCC). We show that calponin within the GCC inhibits the rate of gelsolin mediated nucleation of actin polymerization. The actin-binding function of calponin is ablated within the GCC as the actin-binding site overlaps with one of the gelsolin binding sites. The structure of filaments that result from nucleation by GCC are different to those nucleated by gelsolin alone in that they are longer, loosely bundled and stain heterogeneously with phalloidin. GCC nucleated filaments appear contorted and wrap around each to form the loose bundles.     

A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin

Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (K(d)=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.     

Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin–actin interaction

Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478–485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18–28 and 360–372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the α-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1.     

Two distinct interaction motifs in amphiphysin bind two independent sites on the clathrin terminal domain beta-propeller

During the assembly of clathrin-coated vesicles, many peripheral membrane proteins, including the amphiphysins, use LLDLD-type clathrin-box motifs to interact with the N-terminal beta-propeller domain (TD) of clathrin. The 2.3 A-resolution structure of the clathrin TD in complex with a TLPWDLWTT peptide from amphiphysin 1 delineates a second clathrin-binding motif, PWXXW (the W box), that binds at a site on the TD remote from the clathrin box-binding site. The presence of both sequence motifs within the unstructured region of the amphiphysins allows them to bind more tightly to free TDs than do other endocytic proteins that contain only clathrin-box motifs. This property, along with the propensity of the N-terminal BAR domain to bind curved membranes, will preferentially localize amphiphysin and its partner, dynamin, to the periphery of invaginated clathrin lattices.     

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

The X-ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin-actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH <7, but not at pH 8. Thus the calcium ion trapped in the segment 1-actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.     

Two TPX2-dependent switches control the activity of Aurora A

Aurora A is an important oncogenic kinase for mitotic spindle assembly and a potentially attractive target for human cancers. Its activation could be regulated by ATP cycle and its activator TPX2. To understand the activation mechanism of Aurora A, a series of 20 ns molecular dynamics (MD) simulations were performed on both the wild-type kinase and its mutants. Analyzing the three dynamic trajectories (Aurora A-ATP, Aurora A-ADP, and Aurora A-ADP-TPX2) at the residue level, for the first time we find two TPX2-dependent switches, i.e., switch-1 (Lys-143) and switch-2 (Arg-180), which are tightly associated with Aurora A activation. In the absence of TPX2, Lys-143 exhibits a "closed" state, and becomes hydrogen-bonded to ADP. Once TPX2 binding occurs, switch-1 is forced to "open" the binding site, thus pulling ADP away from Aurora A. Without facilitation of TPX2, switch-2 exits in an "open" conformation which accompanies the outward-flipping movement of P·Thr288 (in an inactive conformation), leading to the crucial phosphothreonine exposed and accessible for deactivation. However, with the binding of TPX2, switch-2 is forced to undergo a "closed" movement, thus capturing P·Thr288 into a buried position and locking its active conformation. Analysis of two Aurora A (K143A and R180A) mutants for the two switches further verifies their functionality and reliability in controlling Aurora activity. Our systems therefore suggest two switches determining Aurora A activation, which are important for the development of aurora kinase inhibitors.     

Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin.

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca(2+)-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxyl-terminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.     

Protein-protein interaction sites in the calcium modulated skeletal muscle troponin complex

The sequence domains that contribute to the surfaces of contact between Troponin-C and the other regulatory protein subunits of skeletal muscle troponin are proposed on the basis of data obtained by proton magnetic resonance and other physicochemical studies on the interaction with Troponin-I of both Troponin-C and its peptide fragments. Marked sequence homology in Troponin-C from various species is found for the residues involved in subunit linkage. The role of the recognition sites is discussed.     

Ufd1 exhibits the AAA-ATPase fold with two distinct ubiquitin interaction sites

Ufd1 mediates ubiquitin fusion degradation by association with Npl4 and Cdc48/p97. The Ufd1-ubiquitin interaction is essential for transfer of substrates to the proteasome. However, the mechanism and specificity of ubiquitin recognition by Ufd1 are poorly understood due to the lack of detailed structural information. Here, we present the solution structure of yeast Ufd1 N domain and show that it has two distinct binding sites for mono- and polyubiquitin. The structure exhibits striking similarities to the Cdc48/p97 N domain. It contains the double-psi beta barrel motif, which is thus identified as a ubiquitin binding domain. Significantly, Ufd1 shows higher affinity toward polyubiquitin than monoubiquitin, attributable to the utilization of separate binding sites with different affinities. Further studies revealed that the Ufd1-ubiquitin interaction involves hydrophobic contacts similar to those in well-characterized ubiquitin binding proteins. Our results provide a structural basis for a previously proposed synergistic binding of polyubiquitin by Cdc48/p97 and Ufd1.     

Two distinct mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex in vivo

We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 "basic patch" is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.     

Exosite binding tethers the macromolecular substrate to the prothrombinase complex and directs cleavage at two spatially distinct sites

The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg(323)-Ile(324), followed by cleavage at Arg(274)-Thr(275). We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDeltaF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaDeltaF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K(m). This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaDeltaF1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIaDeltaF1 to the enzyme. Specific recognition of mIIaDeltaF1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.     

Steady-state nuclear localization of exportin-t involves RanGTP binding and two distinct nuclear pore complex interaction domains

Vertebrate tRNA export receptor exportin-t (Xpo-t) binds to RanGTP and mature tRNAs cooperatively to form a nuclear export complex. Xpo-t shuttles bidirectionally through nuclear pore complexes (NPCs) but is mainly nuclear at steady state. The steady-state distribution of Xpo-t is shown to depend on its interaction with RanGTP. Two distinct Xpo-t NPC interaction domains that bind differentially to peripherally localized nucleoporins in vitro are identified. The N terminus binds to both Nup153 and RanBP2/Nup358 in a RanGTP-dependent manner, while the C terminus binds to CAN/Nup214 independently of Ran. We propose that these interactions increase the concentration of tRNA export complexes and of empty Xpo-t in the vicinity of NPCs and thus increase the efficiency of the Xpo-t transport cycle.     

Ligand-receptor interaction. Klotz-Hunston problem for two classes of binding sites and its solution

The problem of the affinity and quantity determination of two classes of binding sites for ligand-receptor interaction using either Scatchard or Klotz plots was considered. Klotz and Hunston previously solved this problem only for the case of a representation of experimental data using the Scatchard plot. Since their publication, it was the common view that only the use of the Scatchard plot allows solving this problem. However, in some cases, using the Klotz plot is more convenient for a representation of experimental data concerning ligand-receptor interaction, though usually, this plot was used only for the evaluation of receptor affinity with one class of binding sites. In the present paper, it was demonstrated that Klotz plot also could be used for the evaluation affinity and quantity of two classes of binding sites.     

HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites

Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.     

Actin-binding protein amplifies actomyosin contraction,and gelsolin confers calcium control on the direction of contraction

Cross-linking of muscle actin filaments by low concentrations of actin-binding protein reduces the concentration of muscle myosin required for contraction of actin. Gelsolin, a macrophage protein that divides actin filaments in the presence of calcium, inhibits the amplifying effect of actin-binding protein on contraction of actomyosin. In a calcium gradient, the actomyosin gel moves from high to low calcium concentrations, indicating that calcium-controlled lattice formation can impart directionality to the movement of an isotropic actin network.     

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

Alternative splicing of the skeletal muscle Ca_V1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant Ca_V1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (Ca_V1.1 null) myotubes reconstituted with Ca_V1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer Ca_V1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer Ca_V1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, Ca_V1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.     

CD8alphabeta has two distinct binding modes of interaction with peptide-major histocompatibility complex class I

Interaction of CD8 (CD8alphaalpha or CD8alphabeta) with the peptide-major histocompatibility complex (MHC) class I (pMHCI) is critical for the development and function of cytolytic T cells. Although the crystal structure of CD8alphaalpha.pMHCI complex revealed that two symmetric CD8alpha subunits interact with pMHCI asymmetrically, with one subunit engaged in more extensive interaction than the other, the details of the interaction between the CD8alphabeta heterodimer and pMHCI remained unknown. The Ig-like domains of mouse CD8alphabeta and CD8alphaalpha are similar in the size, shape, and surface electrostatic potential of their pMHCI-binding regions, suggesting that their interactions with pMHCI could be very similar. Indeed, we found that the CD8alpha variants CD8alpha(R8A) and CD8alpha(E27A), which were functionally inactive as homodimers, could form an active co-receptor with wild-type (WT) CD8beta as a CD8alpha(R8A)beta or CD8alpha(E27A)beta heterodimer. We also identified CD8beta variants that could form active receptors with WT CD8alpha but not with CD8alpha(R8A). This observation is consistent with the notion that the CD8beta subunit may replace either CD8alpha subunit in CD8alphaalpha.pMHCI complex. In addition, we showed that both anti-CD8alpha and anti-CD8beta antibodies were unable to completely block the co-receptor activity of WT CD8alphabeta. We propose that CD8alphabeta binds to pMHCI in at least two distinguishable orientations.