Self-activation of guanosine triphosphatase activity by oligomerization of the bacterial cell division protein FtsZ.

The essential bacterial cell division protein FtsZ (filamentation temperature-sensitive protein Z) is a distant homologue to the eukaryotic cytoskeletal protein tubulin. We have examined the GTP hydrolytic activity of Escherichia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations greater than 1 microM. At 5 microM FtsZ, we have determined a K(m) of 82 microM GTP and a V(max) of 490 nmol of P(i) min(-1) (mg of protein)(-1). Hydrolysis of GTP requires Mg(2+) and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation experiments show that FtsZ exists as 68% dimer and 13% trimer at 2 microM total protein concentration. Chemical cross-linking of FtsZ also shows that monomer, dimer, trimer, and tetramer species are present at higher (>2 microM) FtsZ concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization. We also monitored the effect of nucleotide and metal ion on the secondary structure of FtsZ; nucleotide yielded no evidence of structural changes in FtsZ, but both Mg(2+) and Ca(2+) had significant effects on secondary structure. Taken together, our results support the hypothesis that Mg(2+)-dependent GTP hydrolysis by FtsZ requires oligomerization of FtsZ. On the basis of these results and structural comparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.

Subunit-subunit interactions in trimeric arginase. Generation of active monomers by mutation of a single amino acid.

The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-A resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554-557). A key feature of this structure is a novel S-shaped oligomerization motif at the carboxyl terminus of the protein that mediates approximately 54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10(5). These monomeric arginase variants are catalytically active, with k(cat)/K(m) values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6-65.5 degrees C, while the corresponding value for the wild-type enzyme is 70 degrees C. The three-dimensional structure of the R308K variant has been determined at 3-A resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase.

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography.

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4.

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.