Harvey J. Sage

The compact conformation of fibronectin is determined by intramolecular ionic interactions.

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin’s compact conformation. Our velocity and equilibrium sedimentation data show that FN2–14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12–14 of one subunit to FN2–3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7–14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7–14 without A does not. Segment FN12–14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12–14, and the presence of A may favor an open conformation by competing with FN2–3 for binding to FN12–14.

The ADAM10 Prodomain Is a Specific Inhibitor of ADAM10 Proteolytic Activity and Inhibits Cellular Shedding Events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-α-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a Ki of 48 nm. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.

Release of Catecholamines and Specific Protein from Adrenal Glands

Rabbit antiserums directed against a purified protein obtained from the catecholamine storage vesicles of the adrenal medulla of the cow were used to study the mechanism of secretion from the adrenal medulla. During secretion evoked by acetylcholine in isolated perfused adrenal glands, catecholamines and the specific intravesicular protein were released in relative amounts comparable to those present in the intact gland. Thus catecholamines are apparently released from the storage vesicles by a process resembling reverse pinocytosis.

Assembly and Molecular Activities of the MutS Tetramer

Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 × 107 m –1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703–710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711–717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.

Kar3 interaction with Cik1 alters motor structure and function

Kar3, a kinesin‐14 motor of Saccharomyces cerevisiae required for mitosis and karyogamy, reportedly interacts with Cik1, a nonmotor protein, via its central, predicted coiled coil. Despite this, neither Kar3 nor Cik1 homodimers have been observed in vivo. Here we show that Kar3 is a dimer in vitro by analytical ultracentrifugation. The motor domains appear as paired particles by rotary‐shadow electron microscopy (EM) and circular dichroism (CD) spectroscopy of the nonmotor region shows characteristics of helical structure, typical of coiled coils. Remarkably, the Kar3/Cik1 nonmotor region shows greater helicity by CD analysis and rotary‐shadow EM reveals a stalk joined to one large or two smaller particles. The highly helical Kar3/Cik1 nonmotor region and visible stalk indicate that dimerization with Cik1 causes structural changes in Kar3. The Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 2–2.4 μm min−1, 2–5‐fold faster than Kar3, and destabilizes microtubules at the lagging ends. Thus, structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo.

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

Background BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to α−tubulin and BtubA to β−tubulin. A 1∶1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament. Methodology/Principal Findings We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1∶1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity. Conclusions An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.

Studies on a phytohemagglutinin from the lentil: V. Binding of Lens culinaris hemagglutinin to lymphocytes and erythrocytes☆

Quantitative studies were performed on the binding of 125I-labelled Lens culinaris hemagglutinin A (LcH-A) to the surfaces of human erythrocytes, human lymphocytes, or rabbit erythrocytes. Human erythrocytes showed 5.8 × 105 surface receptor sites for LcH-A with an apparent association constant, Ka, of 5.9 × 106. Rabbit erythrocytes showed 4.1 × 106 receptor sites with a Ka of 1.7 × 106. Human lymphocytes showed 6.6 × 106 receptor sites with a Ka or 8.1 × 105. In each case the binding of LcH-A to these cells was shown to be reversible and LcH-A could be removed almost totally by washing the cell-LcH-A complex with mannosides. Under conditions used for lymphocyte stimulation, LcH-A was reversibly bound to surface receptors of human lymphocytes; and little, if any, LcH-A was either taken up by the cells or irreversibly bound to the cells during 4 days of culture. During this time extensive lymphocyte stimulation occurred, suggesting that transport of LcH-A into the cell was not a critical part of the stimulation process.

Methionine oxidation of monomeric λ repressor : The denatured state ensemble under nondenaturing conditions

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric λ repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of α‐helical structure in these conformations is detected by far‐UV circular dichroism, and some tertiary interactions are suggested by near‐UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

Separate and Combined Biochemical Activities of the Subunits of a Naturally Split Reverse Gyrase

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single- and double-stranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.

Binding of purified lectins to guinea pig lymphocytes: Studies of the number, binding constant, and distribution of Lens culinaris lectin A and Agaricus bisporus lectin molecules on lymphocyte surfaces

Abstract The surface membrane of guinea pig lymphocytes contains discrete receptors for several different homogeneous lectins. Two very similar lectins from the lentil, LcL-A and LcL-B, bind to the same receptor. Three other lectins, AbL, WGA, and Con A, do not bind detectably to the LcL receptor. AbL binds to another discrete receptor to which none of the other lectin molecules bind. The LcL and AbL receptors are contained on different units, each of which is capable of independent movement on the cell surface. The normal distribution of LcL molecules on the surface is different than the distribution of AbL molecules. However, when lymphocytes are simultaneously incubated with LcL and AbL, the distribution of bound LcL molecules on the cell surface parallels the normal distribution of AbL molecules, which indicates that the movement of AbL molecules and AbL receptors on the surface can strongly influence the movement of LcL and LcL receptors. When lymphocytes are held in close contact by LcL or AbL, the lectin molecules and receptors appear to concentrate at the area of contact between two cells. A model is presented which suggests that the migration of lectins and lectin receptors can occur by a multicellular process.