Browsing by Subject "Mutagenesis, Site-Directed"
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Item Characterization of Internal Dynamics in Vav 1: Method Development, Mutual Coupling and Functional(2009-09-04) Li, Pilong; Rosen, MichaelProtein motions are important to activity, but quantitative relationships between internal dynamics and function are not well understood. The Dbl homology (DH) domain of the proto-oncoprotein and guanine nucleotide exchange factor Vav1 is autoinhibited through interactions between its catalytic surface and a helix from an N-terminal acidic region. Phosphorylation of the helix relieves autoinhibition. Here I show by NMR spectroscopy that the autoinhibited DH domain (AD) exists in equilibrium between a ground state, where the active site is blocked by the inhibitory helix, and an excited state, where the helix is dissociated. Across a series of mutants that differentially sample these states, catalytic activity of the autoinhibited protein and its rate of phosphorylation are linearly dependent on the population of the excited state. Thus, internal dynamics are required for and control both basal activity and the rate of full activation of the autoinhibited DH domain. Vav1 belong to a class of multi-domain signaling proteins exhibit complex behaviors due to cooperative interactions between domains. In many such proteins there is a core regulatory interaction, involving binding of an inhibitory element to the active site of a functional domain like the inhibitory helix to DH in Vav1. The core interaction is cooperatively enhanced by additional intramolecular domain-domain contacts. The physical basis of this cooperativity, and thus the energetic construction of multi-domain systems, is not well understood. Dynamics analysis of AD reveals that the closed and open populations are about 10:1 for the core interaction in isolation. In the full five-domain regulatory fragment of Vav1, interactions between domains outside of the core further bias this inhibitory equilibrium ~10-fold toward the closed state, further suppressing activity. Thus, Vav1 is controlled by two, weakly biasing, but thermodynamically coupled equilibria--an energetic construction that is probably general among multi-domain proteins. The dynamic landscape of AD is composed of two ?s-ms time scale motions: one is the inhibitory helix binding to and dissociating from the DH domain and another is intrinsic to the DH domain. Interestingly relative populations and exchange rates of the second process are altered upon perturbations to the inhibitory helix, suggesting that the two dynamic processes are energetically and kinetically coupled. A strategy has been established to quantify the thermodynamic and kinetic coupling strengths between the two processes via direction parameterization of four-state equilibria using NMR Carr-Purcell-Meiboom-Gill measurement. The coupling strengths between the two dynamic processes in AD are 1.0~1.5 kcal M-1 comparable to the coupling strength between the modulatory interaction and the helix-DH interactions in the full five-domain regulatory fragment of Vav1. The coupling strength is relatively weak consistent with the coupling strengths reported for many other signaling proteins such as Src tyrosine kinase. These findings suggest that weakly coupling may be a common theme in regulatory molecules.Item Structural and Physiologic Determinants of Estrone/Estradiol Metabolism Catalyzed by Human 17beta -Hydroxysteroid Dehydrogenases Types 1 and 2(2006-07-13) Sherbet, Daniel P.; Andersson, StefanThe 17beta -hydroxysteroid dehydrogenases (17beta -HSDs) types 1 and 2 interconvert the weak and potent estrogens estrone and 17beta -estradiol. In intact cells, each enzyme exhibits a strong directional preference that favors either oxidation (17beta -HSD2) or reduction (17beta -HSD1). A positively charged arginine (R38) adjacent to the 2'-phosphate stabilizes NADP(H) binding to 17beta -HSD1 and favors reduction due to the high cytoplasmic NADPH/NADP+ ratio. In contrast, 17beta -HSD2 has a negatively charged glutamate (E116) at the position corresponding to R38 of 17beta -HSD1, which presumably repels the 2'-phosphate of NADP(H) and favors oxidation by harnessing the high cytoplasmic NAD+/NADH ratio. Substitution of a negatively charged aspartate, but not neutral glycine, for R38 of 17beta -HSD1 markedly reduces the affinity for NADP(H) and reverses the directional preference to oxidation in intact cells. We hypothesized that E116 confers oxidative preference to 17beta -HSD2 and that substitution of either a neutral or a positively charged residue for E116 would reverse the directional preference to favor reduction. Mutations E116G, E116R, and the double mutation E116G+N117R failed to attenuate the >95% oxidative preference of 17beta -HSD2 in intact cells. Affinity for all cofactors, as estimated by Km values, were measured for wild-type and mutant 17beta -HSD2 enzymes in yeast microsomes. For wild-type 17beta -HSD2, affinity for NAD(H) is nearly 1000-fold greater than for NADP(H), and the mutant enzymes retain high affinity for NAD(H) yet only slightly better affinity for NADP(H). We conclude that the directional preference of 17beta -HSD1 is principally governed by electrostatic interactions between R38 and the 2'-phosphate of NADP(H), but that the oxidative preference of 17beta -HSD2 is not solely due to E116 in the cofactor-binding domain. These data suggest that the directional preference of 17beta -HSD2 is controlled by other aspects of its cofactor-binding domain, such as the size of the cofactor-binding pocket.