Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.     

Evidence for an inhibitory LIM domain in a rat brain agmatinase-like protein

We recently cloned a rat brain agmatinase-like protein (ALP) whose amino acid sequence greatly differs from other agmatinases and exhibits a LIM-like domain close to its carboxyl terminus. The protein was immunohistochemically detected in the hypothalamic region and hippocampal astrocytes and neurons. We now show that truncated species, lacking the LIM-type domain, retains the dimeric structure of the wild-type protein but exhibits a 10-fold increased k_cat, a 3-fold decreased K_m value for agmatine and altered intrinsic tryptophan fluorescent properties. As expected for a LIM protein, zinc was detected only in the wild-type ALP (∼2 Zn²⁺/monomer). Our proposal is that the LIM domain functions as an autoinhibitory entity and that inhibition is reversed by interaction of the domain with some yet undefined brain protein.     

Structure and dynamics of the human muscle LIM protein

The family of cysteine rich proteins (CRP) comprises three closely homologous members that have been reported to interact with α-actinin. Muscular LIM protein (MLP/CRP3), the skeletal muscle variant, was originally discovered as a positive regulator of myogenesis and is suggested to be part of the stretch sensor of the myofibril through its interaction with telethonin (T-Cap). We determined the structure of both LIM domains of human MLP by nuclear magnetic resonance spectroscopy. We confirm by ¹⁵N relaxation measurements that both LIM domains act as independent units and that the adjacent linker regions are fully flexible. With the published structures of CRP1 and CRP2, the complete family has now been structurally characterized.     

Identification of the key LMO2-binding determinants on Ldb1

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.     

Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

cDNA cloning and mRNA expression of LIM protein gene homologue from the silkworm, Bombyx mori

LIM protein cDNA, from Bombyx mori that contains an open reading frame of 622 bp encoding 94 amino acids, was identified and characterized. The B. mori LIM protein homologue is classified into group 2 LIM proteins that contain glycine-rich LIM domain. B. mori LIM protein mRNA is up-regulated at late embryogenesis and detected in the mid-gut of 5th instar larvae.     

Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, K_v4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca^2 +DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS–DREAM complexes increases upon Ca^2 + association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca^2 +DREAM were determined to be 195 ± 20 μM and 62 ± 4 μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of K_v4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

LIM蛋白家族的研究进展

LIM蛋白是分子结构中含有一个或多个LIM结构域的蛋白质家族,该家族中的蛋白质通过其LIM结构域与某些结构蛋白,激酶,转录调控因子等多种蛋白质相互作用,对某些基因的表达,细胞分化与发育,细胞骨架形成等发挥重要调控作用。本文介绍LIM蛋白家族的分类与功能,LIM蛋白及其与其他蛋白之间的相互作用,以及LIM蛋白在心血管系统中的作用。