B-box1 Domain of MID1 Interacts with the Ube2D1 E2 Enzyme Differently Than RING E3 Ligases.

The MID1 TRIM protein is important for ventral midline development in vertebrates, and mutations of its B-box1 domain result in several birth defects. The B-box1 domain of the human MID1 protein binds two zinc atoms and adopt a similar ßßα-RING structure. This domain is required for the efficient ubiquitination of protein phosphatase 2A, alpha4, and fused kinase. Considering the structural similarity, the MID1 B-box1 domain exhibits mono-autoubiquitination activity, in contrast to poly-autoubiquitination observed for RING E3 ligases. To understand its mechanism of action, the interaction of the B-box1 domain with Ube2D1 (UbcH5a, E2), a preferred E2 ligase, is investigated. Using isothermal titration calorimetry, the MID1 RING and B-box1 domains were observed to have similar binding affinities with the Ube2D1 protein. However, NMR 15N-1H Heteronuclear Single Quantum Coherence titration, 15N relaxation data, and High Ambiguity Driven protein-protein DOCKing (HADDOCK) calculations show the B-box1 domain binding on a surface distinct from where RING domains bind. The novel binding interaction shows the B-box1 domain partially overlapping the noncovalent Ube2D1 and a ubiquitin binding site that is necessary for poly-autoubiquitination activity. The B-box1 domain also displaces the ubiquitin from the Ube2D1 protein. These studies reveal a novel binding interaction between the zinc-binding ßßα-fold B-box1 domain and the Ube2D enzyme family and that this difference in binding, compared to RING E3 ligases, provides a rationale for its auto-monoubiquitination E3 ligase activity.

Anti-Platelet Effect Induced by Iron Oxide Nanoparticles: Correlation with Conformational Change in Fibrinogen.

Iron oxide nanoparticles are developed for various biomedical applications, however, there is limited understanding regarding their effects and toxicity on blood components. The particles traveling in circulation inevitably interact with blood cells and plasma proteins and may interfere with hemostasis. Specifically, this study focuses on the influence of superparamagnetic iron oxide nanoparticles (SPIONs) coated with a biocompatible polymer, polyvinyl alcohol (PVA), on platelet function. Here, engineered SPIONs that are functionalized with various PVA coatings to provide these particles with different surface charges and polymer packing are described. These formulations are assessed for any interference with human platelet functions and coagulation, ex vivo. Positively charged SPIONs induce a significant change in platelet GPIIb-IIIa conformation, indicative of platelet activation at the dose of 500 µg mL-1 . Remarkably, engineered PVA(polyvinyl alcohol)-SPIONs all display a robust dose-dependent anti-platelet effect on platelet aggregation, regardless of the PVA charge and molecular weight. After assessing hypotheses involving SPION-induced steric hindrance in platelet-platelet bridging, as well as protein corona involvement in the antiplatelet effect, the study concludes that the presence of PVA-SPIONs induces fibrinogen conformational change, which correlates with the observed dose-dependent anti-platelet effect.

The structure and behavior of the NA-CATH antimicrobial peptide with liposomes.

Naja atra cathelicidin (NA-CATH) is a 34-amino acid highly cationic peptide identified in Chinese cobras to possess potent toxicity against gram-negative and gram-positive bacteria and low toxicity against host cells. Here, we report the NMR solution structure of the full-length NA-CATH peptide and its interaction with liposomes. The structure shows a well-defined α-helix between residues Phe3 to Lys23, on which one surface is lined by the side-chains of one arginine and 11 lysine residues, while the other side is populated by hydrophobic residues. The last eleven amino acids, which are predominately aromatic and hydrophobic in nature, have no defined structure. NMR data reveal that these residues do not interact with the hydrophobic residues of the helix, indicating that the C-terminal residues have random conformations. Fluorescence requenching experiments, in which liposomes serve as a mimic of the bacterial membranes, result in fluorophore leakage that is consistent with a membrane thinning or transient pore formation mechanism. NMR titration studies of the peptide-liposome interaction reveal that the peptide is in fast exchange with the liposome, consistent with the fluorescent studies. These data indicate that full length NA-CATH possesses a helical segment and unstructured C-terminal tail that disrupts the bilayer to induce leakage and lysing.

The MID1 E3 ligase catalyzes the polyubiquitination of Alpha4 (α4), a regulatory subunit of protein phosphatase 2A (PP2A): novel insights into MID1-mediated regulation of PP2A.

Alpha4 (α4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. α4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of α4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that α4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of α4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in α4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of α4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-α4 interaction and the subsequent polyubiquitination of α4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of α4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of α4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of α4 that is likely to impact the stability and activity level of PP2Ac.

Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding domain: insights into an evolutionarily conserved RING fold.

The B-box type 2 domain is a prominent feature of a large and growing family of RING, B-box, coiled-coil (RBCC) domain-containing proteins and is also present in more than 1500 additional proteins. Most proteins usually contain a single B-box2 domain, although some proteins contain tandem domains consisting of both type 1 and type 2 B-boxes, which actually share little sequence similarity. Recently, we determined the solution structure of B-box1 from MID1, a putative E3 ubiquitin ligase that is mutated in X-linked Opitz G/BBB syndrome, and showed that it adopted a betabetaalpha RING-like fold. Here, we report the tertiary structure of the B-box2 (CHC(D/C)C(2)H(2)) domain from MID1 using multidimensional NMR spectroscopy. This MID1 B-box2 domain consists of a short alpha-helix and a structured loop with two short anti-parallel beta-strands and adopts a tertiary structure similar to the B-box1 and RING structures, even though there is minimal primary sequence similarity between these domains. By mutagenesis, ESI-FTICR and ICP mass spectrometry, we show that the B-box2 domain coordinates two zinc atoms with a 'cross-brace' pattern: one by Cys175, His178, Cys195 and Cys198 and the other by Cys187, Asp190, His204, and His207. Interestingly, this is the first case that an aspartic acid is involved in zinc atom coordination in a zinc-finger domain, although aspartic acid has been shown to coordinate non-catalytic zinc in matrix metalloproteinases. In addition, the finding of a Cys195Phe substitution identified in a patient with X-linked Opitz GBBB syndrome supports the importance of proper zinc coordination for the function of the MID1 B-box2 domain. Notably, however, our structure differs from the only other published B-box2 structure, that from XNF7, which was shown to coordinate one zinc atom. Finally, the similarity in tertiary structures of the B-box2, B-box1 and RING domains suggests these domains have evolved from a common ancestor.

The Diversity of Chemoprotective Glucosinolates in Moringaceae (Moringa spp.).

Glucosinolates (GS) are metabolized to isothiocyanates that may enhance human healthspan by protecting against a variety of chronic diseases. Moringa oleifera, the drumstick tree, produces unique GS but little is known about GS variation within M. oleifera, and even less in the 12 other Moringa species, some of which are very rare. We assess leaf, seed, stem, and leaf gland exudate GS content of 12 of the 13 known Moringa species. We describe 2 previously unidentified GS as major components of 6 species, reporting on the presence of simple alkyl GS in 4 species, which are dominant in M. longituba. We document potent chemoprotective potential in 11 of 12 species, and measure the cytoprotective activity of 6 purified GS in several cell lines. Some of the unique GS rank with the most powerful known inducers of the phase 2 cytoprotective response. Although extracts of most species induced a robust phase 2 cytoprotective response in cultured cells, one was very low (M. longituba), and by far the highest was M. arborea, a very rare and poorly known species. Our results underscore the importance of Moringa as a chemoprotective resource and the need to survey and conserve its interspecific diversity.

Solution structure of the RBCC/TRIM B-box1 domain of human MID1: B-box with a RING.

B-box domains are a defining feature of the tripartite RBCC (RING, B-box, coiled-coil) or TRIM proteins, many of which are E3 ubiquitin ligases. However, little is known about the biological function of B-boxes. In some RBCC/TRIM proteins there is only a single B-box (type 2) domain, while others have both type 1 and type 2 B-box domains in tandem adjacent to their RING domain. These two types of B-boxes share little sequence similarity, except the presence of cysteine and histidine residues: eight in most B-box1 domains and seven in B-box2 domains. We report here the high-resolution solution structure of the first B-box1 domain (from the human RBCC protein, MID1) based on 670 nuclear Overhauser effect (NOE)-derived distance restraints, 12 hydrogen bonds, and 44 dihedral angles. The domain consists of a three-turn alpha-helix, two short beta-strands, and three beta-turns, encompassing Val117 to Pro164, which binds two zinc atoms. One zinc atom is coordinated by cysteine residues 119, 122, 142, 145, while cysteine 134, 137 and histidine 150, 159 coordinate the other. This topology is markedly different from the only other B-box structure reported; that of a type 2 B-box from Xenopus XNF7, which binds a single zinc atom. Of note, the B-box1 structure closely resembles the folds of the RING, ZZ and U-box domains of E3 and E4 ubiquitin enzymes, raising the possibility that the B-box1 domain either has E3 activity itself or enhances the activity of RING type E3 ligases (i.e. confers E4 enzyme activity). The structure of the MID1 B-box1 also reveals two potential protein interaction surfaces. One of these is likely to provide the binding interface for Alpha 4 that is required for the localized turnover of the catalytic subunit of PP2A, the major Ser/Thr phosphatase.

Structural and functional observations of the P151L MID1 mutation reveal alpha4 plays a significant role in X-linked Opitz Syndrome.

Mutations of human MID1 are associated with X-linked Opitz G Syndrome (XLOS), which is characterized by midline birth defects. XLOS-observed mutations within the MID1 B-box1 domain are associated with cleft lip/palate, wide-spaced eyes and hyperspadias. Three of the four XLOS-observed mutations in the B-box1 domain results in unfolding but the structural and functional effects of the P151L mutation is not characterized. Here, we demonstrate that the P151L mutation does not disrupt the overall tertiary structure of the B-box1 domain and the adjacent domains. In fact, MID1 E3 ligase activity is slightly enhanced. However, the P151L mutation disrupted the ability of MID1 to catalyze the poly-ubiquitination of alpha4, a novel regulator of PP2A. This observation is consistent with results observed with the other three structure-destabilizing B-box1 mutations in targeting alpha4 but not PP2A. Alpha4 is shown to bind and sequester the catalytic subunit of PP2A and protect it from MID1-mediated ubiquitination and as a result, an increase in alpha4 can contribute to an increase in PP2A, playing a greater role in midline development during embryogenesis.

FOXP2 variation in great ape populations offers insight into the evolution of communication skills.

The gene coding for the forkhead box protein P2 (FOXP2) is associated with human language disorders. Evolutionary changes in this gene are hypothesized to have contributed to the emergence of speech and language in the human lineage. Although FOXP2 is highly conserved across most mammals, humans differ at two functional amino acid substitutions from chimpanzees, bonobos and gorillas, with an additional fixed substitution found in orangutans. However, FOXP2 has been characterized in only a small number of apes and no publication to date has examined the degree of natural variation in large samples of unrelated great apes. Here, we analyzed the genetic variation in the FOXP2 coding sequence in 63 chimpanzees, 11 bonobos, 48 gorillas, 37 orangutans and 2 gibbons and observed undescribed variation in great apes. We identified two variable polyglutamine microsatellites in chimpanzees and orangutans and found three nonsynonymous single nucleotide polymorphisms, one in chimpanzees, one in gorillas and one in orangutans with derived allele frequencies of 0.01, 0.26 and 0.29, respectively. Structural and functional protein modeling indicate a biochemical effect of the substitution in orangutans, and because of its presence solely in the Sumatran orangutan species, the mutation may be associated with reported population differences in vocalizations.

Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay.

Solution structure of the microtubule-targeting COS domain of MID1.

UNLABELLED: The human MID1 protein is required for the proper development during embryogenesis. Mutations of MID1 are associated with X-linked Opitz G syndrome, characterized by midline anomalies. MID1 associates with the microtubules and functions as an ubiquitin E3 ligase, targeting protein phosphatase 2A for ubiquitin-mediated regulation. The mechanism of microtubule association is not known. Recently, a 60-amino acid region termed the C-terminal subgroup One Signature (COS) box/domain was identified at the C-terminal end of the coiled-coil (CC) domain that facilitates microtubule localization. Insertion of the MID1 COS domain at the C-terminal end of the CC domain of a nonmicrotubule-associated TRIM protein confers microtubule localization. Here, we report the solution structure of the COS domain of MID1. The domain adopts a helix-loop-helix structure in which the N- and C-terminal ends are in close proximity. Hydrophobic residues stabilizing the interaction of the two α-helices form a central hydrophobic core. The loop separating the α-helices is structured, with two of its hydrophobic residues making contact with the central core. On the outer surface, positively charged residues form a distinct basic patch near the termini that we postulate is important for microtubule binding. A model of the structure of the preceding coiled-coil and COS domains (CC-COS) show that the COS domain forms a helical bundle at the C-terminal end of the CC domain similar to the spectrin-like fold observed with some known microtubule-binding proteins. Interestingly, the CC-COS domains bind to microtubules, demonstrating for the first time that MID1 can directly associate with the microtubules. DATABASE: Structural data are available in PDB database under the accession number 5IM8.

Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain.

The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured ß-strand-turn-ß-strand (ß-turn-ß) and the lasso-like loop sub-structures that constitute loop1 of the ßßα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

XLOS-observed mutations of MID1 Bbox1 domain cause domain unfolding.

MID1 catalyzes the ubiquitination of the protein alpha4 and the catalytic subunit of protein phosphatase 2A. Mutations within the MID1 Bbox1 domain are associated with X-linked Opitz G syndrome (XLOS). Our functional assays have shown that mutations of Ala130 to Val or Thr, Cys142 to Ser and Cys145 to Thr completely disrupt the polyubiquitination of alpha4. Using NMR spectroscopy, we characterize the effect of these mutations on the tertiary structure of the Bbox1 domain by itself and in tandem with the Bbox2 domain. The mutation of either Cys142 or Cys145, each of which is involved in coordinating one of the two zinc ions, results in the collapse of signal dispersion in the HSQC spectrum of the Bbox1 domain indicating that the mutant protein structure is unfolded. Each mutation caused the coordination of both zinc ions, which are ∼ 13 Šapart, to be lost. Although Ala130 is not involved in the coordination of a zinc ion, the Ala130Thr mutant Bbox1 domain yields a poorly dispersed HSQC spectrum similar to those of the Cys142Ser and Cys145Thr mutants. Interestingly, neither cysteine mutation affects the structure of the adjacent Bbox2 domain when the two Bbox domains are engineered in their native tandem Bbox1-Bbox2 protein construct. Dynamic light scattering measurements suggest that the mutant Bbox1 domain has an increased propensity to form aggregates compared to the wild type Bbox1 domain. These studies provide insight into the mechanism by which mutations observed in XLOS affect the structure and function of the MID1 Bbox1 domain.

MID1 catalyzes the ubiquitination of protein phosphatase 2A and mutations within its Bbox1 domain disrupt polyubiquitination of alpha4 but not of PP2Ac.

MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.

NMR studies of the C-terminus of alpha4 reveal possible mechanism of its interaction with MID1 and protein phosphatase 2A.

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1.

Detection and characterization of the in vitro e3 ligase activity of the human MID1 protein.

Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains-RING (really interesting new gene), Bbox1, and Bbox2-that have similar ßßα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function.

Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS.

We describe a rapid, simple, and efficient method for recovering glutathione S-transferase (GST)- and His6-tagged maltose binding protein (MBP) fusion proteins from inclusion bodies. Incubation of inclusion bodies with 10% sarkosyl effectively solubilized >95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ratio of Triton X-100 and CHAPS. We demonstrate for the first time that this combination of three detergents significantly improves binding efficiency of GST and GST fusion proteins to gluthathione (GSH) Sepharose.

Structure of the MID1 tandem B-boxes reveals an interaction reminiscent of intermolecular ring heterodimers.

The tripartite motif (TRIM) protein family, defined by N-terminal RING, B-box, and coiled-coil (RBCC) domains, consists of either a single type 2 B-box domain or tandem B-box domains of type 1 and type 2 (B1B2). Here, we report the first structure of the B-box domains in their native tandem orientation. The B-boxes are from Midline-1, a putative ubiquitin E3 ligase that is required for the proteosomal degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). This function of MID1 is facilitated by the direct binding of Alpha4, a regulatory subunit of PP2Ac, to B-box1, while B-box2 appears to influence this interaction. Both B-box1 and B-box2 bind two zinc atoms in a cross-brace motif and adopt a similar betabetaalpha structure reminiscent of the RING, PHD, ZZ, and U-box domains, although they differ from each other and with RING domains in the spacing of their zinc-binding residues. The two B-box domains pack against each other with the interface formed by residues located on the structured loop consisting of the two antiparallel beta-strands. The surface area of the interface is 188 A2 (17% of the total surface). Consistent with the globular structure, the Tm of the tandem B-box domain (59 degrees C) is higher than the individual domains, supporting a stable interaction between the B-box 1 and 2 domains. Notably, the interaction is reminiscent of the interaction of recently determined RING dimers, suggesting the possibility of an evolutionarily conserved role for B-box2 domains in regulating functional RING-type folds.

Mitochondrial and microsomal ferric b5 cytochromes exhibit divergent conformational plasticity in the context of a common fold.

Native-state hydrogen-deuterium exchange (HDX) monitored by NMR spectroscopy has been used to compare conformational plasticity in ferric rat liver outer mitochondrial membrane cytochrome b5 (rOM b5) and ferric bovine liver microsomal cytochrome b5 (bMc b5). Analysis of the data indicated that rOM b5 is the less conformationally flexible protein on the time scale probed by the HDX experiments. The data also suggest a likely contributor to the much higher kinetic barrier for the release of hemin from OM b5s in comparison to Mc b5s, a characteristic that may be to a large extent the source of their divergent functional properties. Specifically, the data indicate that conformational mobility within helices alpha4 and alpha5, which flank the loop harboring axial ligand His63, is considerably more restricted in rOM b5 than in bMc b5. The lower conformational flexibility of alpha4 and alpha5 in rOM b5 can reasonably be attributed to more extensive hydrophobic packing in that region of the protein, arising from two conserved side chain packing motifs in OM cytochrome b5s [Altuve, A., Wang, L., Benson, D. R., and Rivera, M. (2004) Biochem. Biophys. Res. Commun. 314, 602-609].

Mutational, kinetic, and NMR studies of the mechanism of E. coli GDP-mannose mannosyl hydrolase, an unusual Nudix enzyme.

GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-alpha-D-mannose to GDP and the beta-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608). Using the structure and mechanism of MutT, the prototypical Nudix enzyme as a guide, we detected six catalytic residues of GDPMH, three of which were unique to GDPMH, by the kinetic and structural effects of site-specific mutations. Glu-70 (corresponding to Glu-57 in MutT) provides a ligand to the essential divalent cation on the basis of the effects of the E70Q mutation which decreased kcat 10(2.2)-fold, increased the dissociation constant of Mn2+ from the ternary E-Mn2+-GDP complex 3-fold, increased the K(m)Mg2+ 20-fold, and decreased the paramagnetic effect of Mn2+ on 1/T1 of water protons, indicating a change in the coordination sphere of Mn2+. In the E70Q mutant, Gln-70 was shown to be very near the active site metal ion by large paramagnetic effects of Mn2+ on its side chain -NH2 group. With wild-type GDPMH, the effect of pH on log(kcat/K(m)GDPmann) at 37 degrees C showed an ascending limb of unit slope, followed by a plateau yielding a pK(a) of 6.4, which increased to 6.7 +/- 0.1 in the pH dependence of log(kcat). The general base catalyst was identified as a neutral His residue by the DeltaH(ionization) = 7.0 +/- 0.7 kcal/mol, by the increase in pK(a) with ionic strength, and by mutation of each of the four histidine residues of GDPMH to Gln. Only the H124Q mutant showed the loss of the ascending limb in the pH versus log(kcat) rate profile, which was replaced by a weak dependence of rate on hydroxide concentration, as well as an overall 10(3.4)-fold decrease in kcat, indicating His-124 to be the general base, unlike MutT, which uses Glu-53 in this role. The H88Q mutant showed a 10(2.3)-fold decrease in kcat, a 4.4-fold increase in K(m)GDPmann, and no change in the pH versus log(kcat) rate profile, indicating an important but unidentified role of His-88 in catalysis. One and two-dimensional NMR studies permitted the sequence specific assignments of the imidazole HdeltaC, H(epsilon)C, N(delta), and N(epsilon) resonances of the four histidines and defined their protonation states. The pK(a) of His-124 (6.94 +/- 0.04) in the presence of saturating Mg2+ was comparable to the kinetically determined pK(a) at the same temperature (6.40 +/- 0.20). The other three histidines were neutral N(epsilon)H tautomers with pK(a) values below 5.5. Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys. The R52Q mutant decreased kcat 309-fold and increased K(m)GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased K(m)GDPmann 81-fold. The partial rescue of kcat, but not of K(m)GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state. From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group.