David B. McKay

Three-Dimensional Structure of Recoverin, a Calcium Sensor in Vision

Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision. We report here the crystal structure of recombinant unmyristoylated recoverin at 1.9 A resolution. The four EF hands of the protein are arranged in a compact array that contrasts with the dumbbell shape of calmodulin and troponin C. A calcium ion is bound to EF hand 3, while EF hand 2 can bind samarium but not calcium in this crystal form. The other two EF hands have novel structural features that prevent or impair calcium binding. A concave hydrophobic surface formed by EF hands 1 and 2 may participate in the read out of calcium signals by recoverin and its homologs.

Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LCa to stimulate ATP hydrolysis

Uncoating of clathrin-coated vesicles is mediated by the heat shock cognate protein, hsc70, and requires clathrin light chains (LCa and LCb) and ATP hydrolysis. We demonstrate that purified light chains and synthetic peptides derived from their sequences bind hsc70 to stimulate ATP hydrolysis. LCa is more effective than LCb in stimulating hsc70 ATPase and in inhibiting clathrin uncoating by hsc70. These differences correlate with high sequence divergence in the proline- and glycine-rich region (residues 47-71) that forms the hsc70 binding site. For LCa, but not LCb, this region undergoes reversible conformational changes upon perturbation of the ionic strength or the calcium ion concentration. Our results show that LCa is more important for interactions with hsc70 than is LCb and suggest a model in which the LCa conformation regulates coated vesicle uncoating.

Crystallographic Structure of SurA, a Molecular Chaperone that Facilitates Folding of Outer Membrane Porins

The SurA protein facilitates correct folding of outer membrane proteins in gram-negative bacteria. The sequence of Escherichia coli SurA presents four segments, two of which are peptidyl-prolyl isomerases (PPIases); the crystal structure reveals an asymmetric dumbbell, in which the amino-terminal, carboxy-terminal, and first PPIase segments of the sequence form a core structural module, and the second PPIase segment is a satellite domain tethered approximately 30 A from this module. The core module, which is implicated in membrane protein folding, has a novel fold that includes an extended crevice. Crystal contacts show that peptides bind within the crevice, suggesting a model for chaperone activity whereby segments of polypeptide may be repetitively sequestered and released during the membrane protein-folding process.

Lysine 71 of the chaperone protein Hsc70 Is essential for ATP hydrolysis.

It has been proposed that lysine 71 of the bovine 70-kDa heat shock cognate protein might participate in catalysis of ATP hydrolysis by stabilizing an H2O molecule or an OH− ion for nucleophilic attack on the γ-phosphate of the nucleotide (Flaherty, K. M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 12899-12907; Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898). To test this hypothesis, lysine 71 of the ATPase fragment 70-kDa heat shock cognate protein has been mutated to glutamic acid, methionine, and alanine; and the kinetic and structural properties of the mutant proteins have been determined. All three mutant proteins are devoid of measurable ATP hydrolysis activity. Crystal structures of the mutant proteins have been determined to a resolution of 1.7 Å; all three have ATP in the nucleotide binding site. These data identify lysine 71 as a residue that is essential for chemical hydrolysis of ATP.

Definition and spatial location of mouse interleukin-2 residues that interact with its heterotrimeric receptor.

The high affinity receptor for interleukin‐2 (IL‐2) contains three subunits called IL‐2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL‐2 (mIL‐2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL‐2R alpha interaction; three residues, from two separate regions, that were important for IL‐2R beta interaction; and a single residue important for IL‐2R gamma interaction. We built a model mIL‐2 structure based on the homologous human IL‐2 (hIL‐2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL‐2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL‐2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.

Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis.

The leadzyme is a small RNA motif that catalyzes a site-specific, Pb 2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2´-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.

Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase.

The eukaryotic translation initiation factor 4A (elF4A) is a representative of the DEAD-box RNA helicase protein family. We have solved the crystallographic structure of the amino-terminal domain (residues 1-223) of yeast elF4A. The domain is built around a core scaffold, a parallel alpha-beta motif with five beta strands, that is found in other RNA and DNA helicases, as well as in the RecA protein. The amino acid sequence motifs that are conserved within the helicase family are localized to the beta strand-->alpha helix junctions within the core. The core of the amino terminal domain of elF4A is amplified with additional structural elements that differ from those of other helicases. The phosphate binding loop (the Walker A motif) is in an unusual closed conformation. The crystallographic structure reveals specific interactions between amino acid residues of the phosphate binding loop, the DEAD motif, and the SAT motif, whose alteration is known to impair coupling between the ATPase cycle and the RNA unwinding activity of elF4A.

Structure and mechanism of 70-kDa heat-shock-related proteins

Publisher Summary This chapter focuses on the protein chemistry and mechanistic enzymology of the 70-kDa heat-shock-related protein family. Members of this family of proteins are present in cells under normal conditions. Induction of expression of some members in response to heat shock or other forms of cell stress is only a manifestation of an additional facet of the collective functions of the larger family of proteins. A stress-70 protein is localized in the endoplasmic reticulum and is referred as the binding protein. The stress-70 proteins are demonstrated to participate in diverse and seemingly unrelated activities: (1) disassembly of specific macromolecular aggregates, (2) interaction with extended and presumably unfolded polypeptides, and (3) renaturation or reactivation of certain proteins under some conditions. The stress-70 proteins have a common mechanism for transducing free energy of nucleotide hydrolysis into interactions with the protein/polypeptide substrates. The functional organization of stress-70 proteins within their primary, tertiary, and temperature-dependent structural transitions are provided. The enzymatic mechanism of stress-70 proteins involves ATPas mechanism, peptide recognition, coupling of peptide-binding and ATPase activities, and clathrin uncoating reaction.

The metzincin-superfamily of zinc-peptidases.

Over the past three years, the three-dimensional structures of a number of zinc proteinases that share the zinc-binding motif HEXXHXXGXXH have been elucidated. These proteinases comprise astacin, a digestive enzyme from crayfish [1,2,3], adamalysin II [4,5] and atrolysin C [6] from snake venom, the Pseudomonas aeruginosa alkaline proteinase [7] and serralysin from Serratia marcescens proteinase [8], the collagenases from human neutrophils [9,10,11]) and fibroblasts [12,13,14,15], human stromelysin 1 [16; K. Appelt, personal communication] and matrilysin [M. Browner, Keystone Symposia, March 5–12, 1994]. These enzymes represent four different families of zinc peptidases: the astacins [3,17], the bacterial serralysins [18], the adamalysins/reprolysins [19,20], and the matrixins (matrix metalloproteinases, MMPs) [21,22].

Binding of phage-display-selected peptides to the periplasmic chaperone protein SurA mimics binding of unfolded outer membrane proteins

SurA is a periplasmic chaperone protein that facilitates maturation of integral outer membrane proteins (OMPs). Short peptides that bind SurA have previously been characterized. In this work, an enzyme‐linked immunoabsorbent assay‐based competition assay is utilized to demonstrate that binding of such peptides, presented by peptide‐tagged phage, mimics binding of biological substrates. Two representative unfolded OMPs, OmpF and OmpG, bind SurA and a core structural fragment thereof in competition with peptide‐tagged phage, and with the same order‐of‐magnitude affinity as the peptides. Additionally, unfolded OmpF and OmpG bind SurA more tightly than an unfolded water‐soluble protein, while folded proteins have no measurable affinity, demonstrating a specificity of SurA for OMP polypeptides.