Monica Sundd

A hydrogen bond regulates slow motions in ubiquitin by modulating a β-turn flip

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions-the interconversion of a type I into a type II β-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in Cα and Cβ of Ser-37 in tetradecanoyl-ACP. 13C,15N-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C8)- and dodecanoyl (C12)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C14)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pKa value for the carboxylate, ∼1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.

Alcohol-Induced Conformational Transitions in Ervatamin C. An α-Helix to β-Sheet Switchover

Alcohol-induced conformational transitions of erv C, a highly stable cysteine protease, were followed by CD, fluorescence, and activity. At acidic pH, the addition of different alcohols caused two types of conformational transitions. Increasing the concentration of nonfluorinated alkyl alcohols induced a conformational switch from α-helix to β-sheet. Under these conditions, the protein lost its proteolytic activity and tertiary structure. The switch was a sudden one, observed in 50% methanol, 45% ethanol, and 40% propanol. Under similar conditions of pH and concentration, however, glycerol and TFE enhanced the α-helicity of the protein. Methanol-induced denaturation was observed to occur in two stages; the first is the β-sheet state stabilized at low alcohol concentrations, and the other is the β-sheet state with enhanced ellipticity stabilized at high alcohol concentrations. This β-sheet conformation can be attained from the native as well as 6 M GuHCl-denatured state by addition of methanol and exhibits properties different from the native or unfolded state. This state shows loss of tertiary structure and activity, enhanced nonnative secondary structure, noncooperative temperature unfolding, and higher stability toward denaturants as compared to the native state, which are characteristic of the molten globule-like state or O-state, and thus this state may be functioning as an intermediate in the folding pathway of erv C.

Conformational and dynamic changes at the interface contribute to ligand binding by ubiquitin.

Ubiquitin interacts with numerous domains and motifs in its lifetime that vary in structure but bind the same hydrophobic patch. To identify the structural features of ubiquitin that make it an exceptional protein-protein interaction partner, we have studied the interaction of ubiquitin with the signal transducing adaptor molecule-1 ubiquitin interacting motif (UIM) using nuclear magnetic resonance. Our studies bring to light the role of the inherent backbone flexibility of ubiquitin in its interactions with a large array of binding partners, revealed from the changes in C(α) chemical shifts, backbone dynamics, and hydrogen bond lengths upon UIM binding. The crystal structures of ubiquitin complexes lend further support to our findings, underscoring the importance of the unique and flexible hydrogen bond network within ubiquitin and simultaneously providing insights into the nature of the slow motions. Taken together, our studies provide an in-depth view of the molecular changes associated with ligand recognition by ubiquitin.

Crystallization and preliminary X-ray analysis of ervatamin B and C, two thiol proteases from Ervatamia coronaria.

Two highly stable cysteine proteases, ervatamin B (ERV-B) and ervatamin C (ERV-C), purified from the latex of the medicinal plant E. coronaria have been crystallized at room temperature. Crystals of ERV-B and ERV-C diffract to 2.5 and 2.6 A, respectively. The space group is P212121 for the crystals of both proteases with unit-cell parameters a = 47.5, b = 58.8 and c = 68.8 A, and a = 43.8, b = 82.6 and c = 133.1 A, respectively. A self-rotation function for ERV-C indicates a twofold non-crystallographic symmetry relating the two molecules in the asymmetric unit.

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

Backbone and side chain 1H, 15N & 13C chemical shift assignments of the holo-acyl carrier protein (ACP) of Leishmania major

Acyl carrier protein (ACP) is a small acidic protein, an important cofactor involved in fatty acid biosynthesis. Its main function is to protect the growing acyl chain from the hydrophilic environment during fatty acid biosynthesis and simultaneously, present it to the active site of fatty acid pathway enzymes, liable for its elongation. The ACP molecule is expressed as apo-ACP (inactive) and is post-transitionally modified to the holo form (active) by the enzyme holo ACP synthase (ACPS). Here we report the complete backbone and side chain chemical shift assignments of the holo-ACP molecule of Leishmania major.

Backbone chemical shift assignments of the glycine cleavage complex H protein of Escherichia coli

Glycine cleavage complex H protein (GcvH) is one of the four components that form the glycine cleavage complex (GCS), essential for the synthesis of C1 (one-carbon units) for cell metabolism, by the oxidative cleavage of glycine. The activity of this complex is induced in the presence of exogenous glycine, and is repressed by purines. GCS, in cooperation with GCA (serine hydroxymethyltransferase) regulates the endogenous levels of glycine and C1 units in the cell. GcvH, the lipoamide containing component of the complex, plays an indispensable role in this reaction, as its prosthetic group shuttles between the active site of the three other components of the GCS complex sequentially. In environments rich in exogenous lipoic acid, GcvH is converted to lipoyl-GcvH by Lipoate protein ligase (LplA), by the salvage pathway. When exogenous lipoic acid is deficient, it is post-translationally modified to lipoyl-GcvH by the consecutive action of two enzymes, (a) Lipoate protein ligase B (LipB) and (b) Lipoyl synthase (LipA). Although, the crystal structure has been determined for Escherichia coli GcvH, no information exists for its interaction with LipB or LipA. Therefore, we plan to study its interactions with the aforementioned enzymes. As a first step, we have carried out the complete backbone chemical shift assignments of the E. coli glycine cleavage complex H protein in its apo-form, as well as its C8- intermediate.

Grb2 carboxyl-terminal SH3 domain can bivalently associate with two ligands, in an SH3 dependent manner

Src homology domain containing leukocyte protein of 65 kDa (SLP65), the growth factor receptor binding protein 2 (Grb2), and the guanine nucleotide exchange factor for the Rho family GTPases (Vav), self associate in unstimulated B cells as components of the preformed B cell receptor transducer module, in an SH3-dependent manner. The complex enables the B cell to promptly respond to BCR aggregation, resulting in signal amplification. It also facilitates Vav translocation to the membrane rafts, for activation. Here we uncover the molecular mechanism by which the complex may be formed in the B cell. The C-terminal SH3 domain (SH3C) of Grb2 bivalently interacts with the atypical non-PxxP proline rich region of SLP65, and the N-terminal SH3 domain (SH3N) of Vav, both the interactions crucial for the proper functioning of the B cell. Most surprisingly, the two ligands bind the same ligand binding site on the surface of Grb2 SH3C. Addition of SLP65 peptide to the Grb2-Vav complex abrogates the interaction completely, displacing Vav. However, the addition of Vav SH3N to the SLP65-Grb2 binary complex, results in a trimeric complex. Extrapolating these results to the in vivo conditions, Grb2 should bind the SLP65 transducer module first, and then Vav should associate.