The low density lipoprotein receptor-related protein (LRP) 1 is a large endocytic receptor containing a 515-kDa heavy chain to which ligands bind and a non-covalently associated 85-kDa light chain containing a transmembrane and cytoplasmic domain (for review see Ref. 1). LRP is one of 12 or more receptors that make up the LDL receptor superfamily and is essential for embryonic development in mice (2). A remarkable feature of LRP is its ability to bind and mediate the internalization of a diverse array of ligands, including proteinases (3, 4), proteinase-inhibitor complexes (5, 6), and lipoproteins (7). After binding to the LRP, the ligands are transported into endosomes where they uncouple in the reduced pH environment and are sorted to lysosomes for degradation. LRP recycles back to the cell surface where it is once again available to bind ligands.Recent studies indicate that in addition to their cargo transport function, certain LDL receptor family members also participate in signaling pathways. For example, the very low density lipoprotein receptor and apoE receptor 2 both participate in a signal transduction pathways mediated by reelin (8 -10). Reelin is secreted by Cajal-Retzius cell in the outermost layer of the cerebral cortex and controls the final position of neurons that migrate from the ventricular zone. Binding of reelin to either the very low density lipoprotein receptor or apoE receptor 2 induces tyrosine phosphorylation of disabled-1 (Dab1) (9, 10), an adaptor protein that interacts with the cytoplasmic domains of LDL receptor family members (11, 12) and functions in tyrosine kinase signaling pathways.In the case of LRP, accumulating evidence suggests a prominent but undefined role for this receptor in regulating cell physiology by facilitating signal transduction pathways. For example, LRP has been implicated as a component of the receptor complex for midkine (13), a heparin binding growth factor with migration-promoting and survival-promoting activities. Another LRP ligand, tissue type plasminogen activator, promotes late phase long term potentiation (14), and this activity appears to require its association with LRP (15). Finally, the binding of activated ␣ 2 M (␣ 2 M*) to LRP mediates calcium influx in neurons in a process that also involves N-methyl-D-

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptorrelated protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors. Apolipoprotein E (apoE) is a 34 kDa protein that plays an important role in lipoprotein metabolism by association with lipoprotein particles and with members of the LDL receptor family (1, 2). ApoE contains a 22 kDa N-terminal domain (residues 1-191) that is recognized by receptors and a 10 kDa C-terminal domain (residues 222-299) that has high affinity for lipid and is responsible for the association of apoE with lipoproteins (3, 4). Three major isoforms of apoE exist in the population and differ by cysteine and arginine at residues 112 and 158. The most common isoform, apoE3, contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine at both positions and apoE4 contains arginine at both positions (5). These substitutions have important biological consequences. First, the various apoE isoforms are differentially recognized by the LDL receptor. Thus, apoE3 and apoE4 readily bind to the LDL receptor, whereas apoE2 binds poorly to the LDL receptor and is associated with type III hyperlipidemia (6). Second, the APOE-4 allele is associated with the common late-onset familial and sporadic forms of Alzheimer's disease (AD) (7,8). The biochemical mechanism by which the APOE-4 allele increases the risk of AD is unknown, but several possibilities have been proposed (9-11), including differential functions of apoE isoforms upon interaction with members of the LDL receptor family (9).The LDL receptor family includes the LDL receptor, the LDL receptor-related protein (LRP), LRP1b, megalin (or LRP-2), the VLDL receptor, and apoE receptor 2 (for Abbreviations: AD, Alzheimer's disease; apoE, apolipoprotein E; LRP, low density lipoprotein...

The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.

Transcription factor p63, a p53 family member, plays a role in epithelial cell development, cell cycle arrest, apoptosis, and tumorigenesis. Point mutations, primarily in the DNA binding domain (p63DBD), lead to malformation syndromes. To gain insight into differences between p63 and p53 and the impact of mutations on the structure, we have determined two crystal structures of p63DBD in complex with A/T-rich response elements. One complex contains a 10-bp DNA half-site response element (5′AAACATGT-TT3′) and the other contains a 22-bp DNA full response element with a 2-bp spacer between two half-sites (5′AAACATGTTTTAAAA-CATGTTT3′). In both structures, each half-site binds a p63DBD dimer. The two p63DBD dimers do not interact in the presence of the DNA spacer, whereas they interact with one another in the p63DBD/10-bp complex where the DNA simulates a full response element by packing end-to-end. A unique dimer-dimer interaction involves a variable loop region, which differs in length and sequence from the counterpart loop of p53DBD. The DNA trajectories in both structures assume superhelical conformations. Surface plasmon resonance studies of p63DBD/DNA binding yielded K d ¼ 11.7 μM for a continuous full response element, whereas binding was undetectable with the 22-bp DNA, suggesting an important contribution of a p63DBD interdimer interface to binding and establishing that p63DBD affinity to the response element is approximately 1,000-fold lower than that of p53DBD. Analyses of the structural consequences of p63DBD mutations that cause developmental defects show that, although some mutations affect DNA binding directly, the majority affects protein stability.T he transcription factor p63 controls the development and morphogenesis of epithelial tissues (1-3). Mutations in the gene cause limb and orofacial defects, many of which occur in the DNA binding domain (4). Overexpression of the isoforms lacking the transactivation domain promotes tumorigenesis in some cancers (5-7). p63 is a member of a transcription factor family that also includes the p53 tumor suppressor and p73 (8). These are multidomain proteins that contain an N-terminal transactivation (TA) domain, a DNA binding domain (DBD), and a tetramerization domain (TD). Additionally, p63 and p73, but not p53, contain at their C termini a domain known to interact with other proteins, the sterile alpha motif (SAM), followed by a transcription inhibitory domain (TID) (8, 9). Alternative promoters of the p53, p63, and p73 genes yield the respective gene products with truncated TA domains. Several alternative splicing sites at the 3′ end of both the p63 and p73 transcripts truncate the SAM and TID domains and result in additional isoforms (10). In contrast to p53-null mice, which are developmentally normal, p63-null mice revealed a complex relationship between the gene and development, with severe deleterious effects (2, 3).p53 family members function as tetramers. Their response elements contain two tandem repeats of 10-bp half-sites without a spacer or with a s...

Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+). Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex. Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.