(2003) Nat. Struct. Biol. 10, 757-763). The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.A recent description of a rare human allele of porphobilinogen synthase (PBGS) 1 revealed that the enzyme can exist as two alternate quaternary structures wherein the oligomeric state is dictated by distinctly different structures for their component crystallographic asymmetric unit (1). The monomer is an ␣␤-barrel protein with a 23-residue N-terminal arm. The octameric form is comprised of four "hugging dimers" (Fig. 1a), whereas the hexameric form is comprised of three "detached dimers" (Fig. 1b). In both oligomeric assemblies there are invariant barrel-to-barrel subunit interactions within the dimers and quasiequivalent arm-to-bottom-of-barrel subunit interactions between adjacent dimers (Fig. 1). However, the octamer has a unique "hugging" arm-to-barrel interaction that is not seen in the hexamer.Preparations of heterologously expressed wild type human PBGS predominantly contain octamer, but there is a small propensity to adopt the hexameric structure (1). In contrast, heterologous expression of the rare human variant, F12L, yields protein that purifies as the hexamer and does not readily take on the octameric structure. The homo-octameric wild type and homohexameric F12L forms of human PBGS exhibit dramatic differences in pH activity profile and in the kinetic K m values (Table I) (1) despite the fact that amino acid residue 12 does not interact directly with active site residues in either quaternary structure isoform (Fig. 1). However, the arm-tobarrel interface found in the hugging dimer provides substantial stabilization for the solvent-accessible face of the ␣␤-barrel wherein lies the active site. This region of the protein, which is disordered in the hexamer, includes an ϳ11-amino acid stretch that normally serves to gate the active site from solvent, also called the active site lid. With the active site more solventaccessible in the hexamer, a high external pH is required to drive the Schiff base formation that is necessary for catalysis (2); this contributes to the high pH required for F12L activity. The decreased affinity of F12L for substrate is attributed to the loss of interactions between the destabilized active site lid and the carboxyl moiety of the outermost substrate (3).Coexpression of wild type and F12L (WTϩF12L...

Enzymes that regulate their activity by modulating an equilibrium of alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) constitute a recently described mode of allostery. The oligomeric equilibrium for porphobilinogen synthase (PBGS) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. A phylogenetically diverse allosteric site specific to hexamers is proposed as an inhibitor binding site. Inhibitor binding is predicted to draw the oligomeric equilibrium toward the low-activity hexamer. In silico docking enriched a selection from a small-molecule library for compounds predicted to bind to this allosteric site. In vitro testing of selected compounds identified one compound whose inhibition mechanism is species-specific conversion of PBGS octamers to hexamers. We propose that this strategy for inhibitor discovery can be applied to other proteins that use the morpheein model for allosteric regulation.

Solid tumors develop abnormally at spatial and temporal scales, giving rise to biophysical barriers that impact anti-tumor chemotherapy. This may increase the expenditure and time for conventional drug pharmacokinetic and pharmacodynamic studies. In order to facilitate drug discovery, we propose a mathematical model that couples three-dimensional tumor growth and angiogenesis to simulate tumor progression for chemotherapy evaluation. This application-oriented model incorporates complex dynamical processes including cell- and vascular-mediated interstitial pressure, mass transport, angiogenesis, cell proliferation, and vessel maturation to model tumor progression through multiple stages including tumor initiation, avascular growth, and transition from avascular to vascular growth. Compared to pure mechanistic models, the proposed empirical methods are not only easy to conduct but can provide realistic predictions and calculations. A series of computational simulations were conducted to demonstrate the advantages of the proposed comprehensive model. The computational simulation results suggest that solid tumor geometry is related to the interstitial pressure, such that tumors with high interstitial pressure are more likely to develop dendritic structures than those with low interstitial pressure.

Evidence is building to support the notion that the porphobilinogen synthase (PBGS 2 ; EC 4.2.1.24) family of enzymes can exist as an equilibrium of quaternary structure isoforms, denoted morpheeins (1-3). Morpheeins comprise an equilibrium ensemble of protein structures wherein a protein monomer can exist in more than one conformation, and each monomer conformation dictates a functionally different quaternary structure of finite multiplicity. Morpheeins have been proposed to provide a structural foundation for allosteric regulation, cooperativity, and hysteresis in some proteins (2). As such, the energetic difference between morpheeins of a given protein must be small. The propensity of PBGS to assume various morpheein structures and the rates of PBGS morpheein interconversion are highly species-dependent. The stable morpheeins of human PBGS are the octamer, found for the wild-type protein, and the hexamer, first seen for the naturally occurring mutation F12L (1). Coexpression of human wild-type PBGS and F12L generates a population of PBGS proteins composed of hetero-octamers and heterohexamers, each of which contains a mixture of Phe 12 -and Leu 12 -containing chains (1). The structure and composition of these hetero-oligomers are stable during storage, but the molecular motions resulting from catalysis favor formation of the octamer with an accompanying disproportionation of Phe 12 -containing chains into the octamer (3).The remaining hexamer has an increased proportion of Leu 12 -containing chains (3). The physical basis for the thermodynamic propensity of Leu 12 -containing chains to form the hexamer remains unclear, but examination of the structure of human PBGS suggests that other single amino acid mutations might affect the folding and assembly of the protein to favor structures other than the octamer. In this study, we report on alterations of two amino acids (Arg 240 and Trp 19 ) that were chosen based on an analysis of the structures and subunit interactions seen in human octameric and hexameric PBGS, for which the assemblies are shown in Fig. 1. Each human PBGS subunit is composed of a 306-amino acid TIM-like ␣␤-barrel and a 24-amino acid N-terminal arm. The conformational difference between the monomer that assembles into the octamer and the monomer that assembles into the hexamer is a dramatic reorientation of the arm with respect to the barrel; for the F12L hexamer, this reorientation occurs at Thr 23 . In both the octameric and hexameric assemblies, two monomers come together to form a dimer with a conserved barrel-barrel interface. The dimer that assembles into the octamer is called a hugging dimer (Fig. 1a), whereas the dimer that assembles into the hexamer is called a detached dimer (Fig. 1b) (1). The difference between these two dimers is the presence or absence of a "hugging" interaction between the N-terminal arm of one subunit and the ␣␤-barrel of the adjacent subunit of the dimer. The "arm-hugging-barrel" interaction of PBGSs from plants, Archaea, and most Bacteria is stabilized by an all...

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.

A secreted counting factor (CF), regulates the size of Dictyostelium discoideum fruiting bodies in part by regulating cell-cell adhesion. Aggregation and the expression of adhesion molecules are mediated by relayed pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. A 1-min exposure of cells to purified CF increases the cAMP-induced cAMP pulse. CF does not affect the cAMP receptor or its interaction with its associated G proteins or the translocation of the cytosolic regulator of adenylyl cyclase to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, whereas reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The results suggest that regulating cell-cell adhesion, the cGMP pulse size, or the cAMP pulse size can control group size and that CF regulates all three of these independently.

Little is known about how a morphogenetic rearrangement of a tissue is affected by individual cells. Starving Dictyostelium discoideum cells aggregate to form dendritic streams, which then break up into groups of Ϸ2 ؋ 10 4 cells. Cell number is sensed at this developmental stage by using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF indicates that there is a large number of cells, which then causes the aggregation stream to break up. Computer simulations indicated that stream breakup could be caused by CF decreasing cell-cell adhesion and͞or increasing cell motility, and we observed that CF does indeed decrease cell-cell adhesion. We find here that CF increases cell motility. In Dictyostelium, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and the ABP-120 actin-crosslinking protein. Partially inhibiting motility by using drugs that interfere with actin polymerization reduces stream dissipation, resulting in fewer stream breaks and thus larger groups. CF also potentiates the phosphorylation and redistribution of myosin while repressing its basal level of assembly. The computer simulations indicated that a narrower distribution of group sizes results when a secreted factor modulates both adhesion and motility. CF thus seems to induce the morphogenesis of streams into evenly sized groups by increasing actin polymerization, ABP-120 levels, and myosin phosphorylation and decreasing adhesion and myosin polymerization.

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.