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The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of Ϸ550 Å 3 , which is smaller than that determined from the structure of deprenyl-inhibited hMAO B (Ϸ700 Å 3 ) but larger than that of rat MAO A (Ϸ450 Å 3 ). An important component of the active site structure of hMAO A is the loop conformation of residues 210 -216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 3 Lys mutation that is specific flavin ͉ neurotransmitter ͉ membrane protein ͉ antidepressant target H uman monoamine oxidase A (hMAO A) is an outer mitochondrial membrane-bound flavoenzyme that catalyzes the oxidation of the neurotransmitters serotonin, dopamine, and norepinephrine. Recent studies have demonstrated that a deficiency or low level of expression of this enzyme results in a phenotype of aggressive behavior (1, 2). The elucidation of the 3D structures of human MAO B (hMAO B) (3, 4) (72% sequence identity with hMAO A) and of rat MAO A (rMAO A) (5) (92% sequence identity with hMAO A with no insertions or deletions) has provided insights into the structure and mechanism of these pharmacologically important enzymes. There are several functional properties of hMAO A that differentiate it from rMAO A, despite their high level of sequence identity. hMAO A has been shown to exhibit a 10-fold lower affinity (IC 50 ) than rMAO A for the specific irreversible inhibitor clorgyline (6). Comparisons of the influence of a Phe-208 3 Ile mutation on MAO A from human (7) and rat (8) also show differential effects on activities and sensitivities to irreversible inhibition. Functional differences between hMAO A and rMAO A have been implicated in comparison with their respective sensitivities to phentermine inhibition (9). These differences in properties between hMAO A and rMAO A suggest structural differences exist for these two enzymes.With the development of a high-level expression system for hMAO A in our laboratory (10) and successes with the structural elucidation of hMAO B (3, 4), a collaborative program was established to elucidate the structure of hMAO A by x-ray crystallography. Here, we report the structures of two hMAO A crystal forms and demonstrate structural differences between hMAO A and rMAO A as well as hMAO B. Our data indicate that the considerable literature on MAO A-inhibitor development by using rat models m...

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.

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