doi: 10.1002/bmb.20211.
Expanding the Concepts in Protein Structure-Function Relationships and Enzyme Kinetics: Teaching using Morpheeins
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- PMID:19578473
- PMCID: PMC2575429
- DOI: 10.1002/bmb.20211
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Expanding the Concepts in Protein Structure-Function Relationships and Enzyme Kinetics: Teaching using Morpheeins
Sarah H Lawrence et al. Biochem Mol Biol Educ.2008.
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Abstract
A morpheein is a homo-oligomeric protein that can exist as an ensemble of physiologically significant and functionally different alternate quaternary assemblies. Morpheeins exist in nature and utilize conformational equilibria between different tertiary structures to form distinct oligomers as a means of regulating their function. Notably, alternate morpheeins are not misfolded forms of a protein; they are differently assembled native states that contain alternate subunit conformations. Transitions between alternate morpheein assemblies involve oligomer dissociation, conformational change in the dissociated state, and reassembly to a different oligomer. These transitions occur in response to the protein's environment, e.g., effector molecules, and represent a new model of allosteric regulation. The unique features of morpheeins are being revealed through detailed characterization of the prototype enzyme, porphobilinogen synthase, which exists in a dynamic equilibrium of a high activity octamer, a low activity hexamer, and two dimer conformations. Morpheeins are likely far more common than previously appreciated. There are, however, both intellectual and experimental barriers to recognizing proteins as morpheeins. These barriers derive from the way we were taught and continue to teach about protein folding, protein purification, protein structure-function relationships, and enzyme kinetics. This article explores some of these limitations and encourages incorporation of morpheeins into both introductory and advanced biochemistry classes.
Figures
Four possible conformations of the monomer are shown in cyan (A), green (B), lilac (C), and pink (D). A protein that functions as a morpheein need only adopt two of these monomer conformations. Following a “rule of engagement” where oligomerization must occur by an association of a dashed line with a thick line, the subunits can self-engage to monomers of A or oligomerize to dimers of B, trimers of C, or tetramers of D. Monomer A is comparable to what have previously been termed “auto-inhibited” conformations of some proteins that are active as dimers.
The similarities and differences of the classic protein folding paradigm (left),versus the morpheein model (right).
(A) The main quaternary assemblies of human porphobilinogen synthase are a low-activity hexamer (light and dark blue) and a high-activity octamer (light and dark pink). Each assembly is shown as viewed from the top with the subunits shown as spheres.(B) The morpheein equilibrium of human PBGS involves the hexamer, the octamer and two structurally distinct dimer conformations. The dissociated hexamer is shown as a detached dimer, which is in equilibrium with a hugging dimer, which associates to the octamer. The dimers are shown as cartoons. The detached and hugging dimers are shown in the context of the hexamer and octamer, respectively, with the remaining subunits shown as spheres.(C) The detached dimer (light and dark blue) superimposes almost perfectly with the hugging dimer (shades of pink), with the exception of the N-terminal arm (24 of a total 330 amino acids); the orientation of this arm guides higher order oligomerization. Note that the illustrated dimer conformations correspond to the asymmetric units of crystal structures PDB codes 1PV8 and 1E51 (hexamer and octamer respectively); we have presented alternative dimeric assemblies as the solution structures of the dimer configurations [12].
(A) In a classic enzyme-catalyzed reaction, the catalytic rate increases as a hyperbolic function of substrate concentration (grey squares and dashed line) system. In the event that two kinetically distinct forms of an enzyme coexist, the data (black circles) cannot be fit to a single hyperbola (dotted black line). Instead, the data fit to a double hyperbola (solid line), where the sum of two Michaelis-Menten equations describes the contributions from both species.(B). In a classic enzyme-catalyzed reaction (dashed grey line) the product in creases linearly with time as long as substrate is saturating. For an enzyme that exists as an equilibrium of morpheein forms (solid black line), a lag time can reflect an inactive form dissociating, equilibrating, and re-associating into a more active form in the presence of substrate. Alternatively, in the presence of an inhibitor, a rapid initial rate can slowly approach a final steady state rate as the ensemble of morpheein forms reequilibrates to the less active assembly (not shown).(C) The activity per enzyme unit (specific activity) is a fixed property that does not vary with enzyme concentration for standard enzymes (dashed grey line). In a morpheein system in which a higher protein concentration favors a more active assembly, the specific activity increases as a function of enzyme concentration (solid black line). Alternatively, if the lower stoichiometry assembly is more active, one might see an inverse relationship between enzyme concentration and specific activity (not shown).
(A) In the Monod, Wyman, Changeux (MWC, or concerted) model of allostery, each oligomer exists in the inactive (composed of round subunits) or active (composed of square subunits) states. The oligomers are in equilibrium with each other, and binding of an allosteric activator (shown as jagged black squares), draws the equilibrium towards the active state.(B) In the Koshland, Nemethy, Filmer (KNF, or sequential) model of allostery, the individual subunits of the oligomers exist in the inactive (round) or active (square) state. Binding of an allosteric activator to a subunit within an oligomer induces a conformational change of an adjacent subunit to the active state.(C) In the example of the morpheein model, allosteric control involves dissociation of the oligomers. The inactive form is a trimer of pie-wedge subunits, the active form is a tetramer of square subunits; the trimers, pie-wedges, squares, and tetramers all coexist in equilibrium. The allosteric activator binds to the square subunits and draws the equilibrium towards the active tetramer. For example, in the case of plant PBGS, the allosteric activator is a magnesium ion [11].
(A) The insulin receptor is composed of monomers in its inactive state. The binding site for insulin exists at the dimerization interface, and the binding of a single insulin molecule tethers the monomers into a dimer, turning on the signal. The insulin receptor does not function as a morpheein.(B) The EGF receptor also exists as monomers in the inactive state, but the monomers equilibrate between a closed (auto-inhibited) structural form that cannot oligomerize, and an open structural form that can. Dimerization is mediated by insertion of the dimerization arm (shown in red) from one monomer into a pocket on the other monomer. This pocket only exists in the open structural form; in the auto-inhibited form, the dimerization arm self-associates with its own monomer. The receptor binding site is distant from the oligomeric interface. Binding of EGF stabilizes the open form and draws the oligomeric equilibrium to the dimer, turning on the signal. The EGF receptor functions as a morpheein.
For a morpheein that exists as an inactive trimer formed of pie-wedge subunits and an active tetramer formed of square subunits, an inhibitor (yellow wedge) would function by binding to the pie-wedges or trimers, and drawing the equilibrium towards the inactive form.
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