The importance of being dimeric

Mei, Giampiero; Venere, Almerinda Di; Rosato, Nicola; Finazzi‐Agrò, Alessandro

doi:10.1111/j.1432-1033.2004.04407.x

Cited by 100 publications

(100 citation statements)

References 68 publications

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“…3). The structure conformation that is responsible for the stability of a protein has been reported in many case studies (Mei et al, 2005). According to the experimental results described above, the human SULT1A1 dimer might be stabilized by …”

Section: Discussionmentioning

confidence: 76%

Dimerization Is Responsible for the Structural Stability of Human Sulfotransferase 1A1

Lu¹,

Chiang²,

Chen³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Cytosolic sulfotransferases (SULTs) are responsible for the metabolism of a variety of drugs, xenobiotics, and endogenous compounds. Most cytosolic SULTs are found to be homodimers. However, transformation between monomeric and dimeric SULTs can be achieved by a single amino acid mutation. The importance of quaternary structure for cytosolic sulfotransferase was investigated using recombinant human SULT1A1, a homodimer, and its monomeric mutant (V270E). The differences between dimeric and monomeric SULT1A1 were examined by size-exclusion liquid chromatography, enzyme kinetics, substrate binding affinity, thermal inactivation, conformational stability, and circular dichroism. Variations, especially on their secondary structures and stability, between homodimer and monomer of human SULT1A1 were observed. It was found that the active site of SULT1A1 was not significantly perturbed after the change of its quaternary structure according to SULT1A1 kinetics and substrate binding affinity. However, the stability of monomeric SULT1A1 is significantly decreased. We proposed that the importance of human SULT1A1 as a homodimer was to maintain its structural stability, and the change of secondary structure was responsible for alternating its quaternary structure.

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Section: Discussionmentioning

confidence: 76%

Dimerization Is Responsible for the Structural Stability of Human Sulfotransferase 1A1

Lu¹,

Chiang²,

Chen³

et al. 2009

Drug Metab Dispos

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“…For example, dimeric proteins, which represent Ϸ8% of all protein structures (37), play essential roles in a number of biological systems. In addition, the same methods should be useful in designing protein-protein pairs with novel specificity that could be used in the synthesis and study of specific protein signaling events.…”

Section: Predicted Vs Experimental Stabilitiesmentioning

confidence: 99%

Specificity versus stability in computational protein design

Bolon

Grant

Baker

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

135

123

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Protein-protein interactions can be designed computationally by using positive strategies that maximize the stability of the desired structure and͞or by negative strategies that seek to destabilize competing states. Here, we compare the efficacy of these methods in reengineering a protein homodimer into a heterodimer. The stability-design protein (positive design only) was experimentally more stable than the specificity-design heterodimer (positive and negative design). By contrast, only the specificity-design protein assembled as a homogenous heterodimer in solution, whereas the stability-design protein formed a mixture of homodimer and heterodimer species. The experimental stabilities of the engineered proteins correlated roughly with their calculated stabilities, and the crystal structure of the specificity-design heterodimer showed most of the predicted side-chain packing interactions and a mainchain conformation indistinguishable from the wild-type structure. These results indicate that the design simulations capture important features of both stability and structure and demonstrate that negative design can be critical for attaining specificity when competing states are close in structure space.adaptor protein ͉ protein engineering ͉ SspB H ighly specific recognition lies at the core of most cellular processes. The interaction of two partner proteins in a crowded intracellular environment depends on the equilibrium stability of the complex, which is determined by affinity and concentration, but is also controlled by the competing binding of either partner to other cellular macromolecules. Efficient protein recognition requires both stability and specificity. In natural systems, both parameters are subject to evolutionary optimization. Likewise in engineered systems, stability can be targeted for maximization, and known competing states or interactions can be minimized through the use of negative design. The biophysical tradeoff between stability and specificity in molecular recognition is a fascinating problem that has important implications in the development of practical tools for synthesizing and dissecting biological systems.Focusing on stability without explicit consideration of specificity has resulted in impressive protein-engineering feats, including full-sequence design of a zinc-finger protein that folds in the absence of metal (1), introduction of catalytic activity into previously inert protein scaffolds (2, 3), and the creation of a novel protein fold (4). These successes relied on positive design only. Positive design maximizes favorable interactions in the target conformation. Negative design, by contrast, maximizes unfavorable interactions in competing states and requires modeling of each unwanted conformation (5, 6).Protein-protein interactions have been successfully reengineered to alter binding specificity both by using and ignoring negative design (7-15). How is success possible in the absence of negative design? One possibility is that most changes that optimize the stability of a targe...

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“…For instance, their quaternary structure can be regarded as the simplest case of protein-protein interaction. Moreover, proteins can gain new structural and functional properties upon association of subunits, which are often unstable when separated (23,24). Fully folded monomeric intermediates are in fact an exception rather than a rule; in most cases they are only partially folded prior to association, resembling the so-called molten globule state, because of their loose tertiary structure.…”

Section: Faah Stability Is Strongly Dependent On Its Quaternarymentioning

confidence: 99%

Closing the Gate to the Active Site

Mei

Venere

Gasperi

et al. 2007

Journal of Biological Chemistry

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Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of ≈30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of ≈11 kcal/mol that accounted for ≈80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (⌬G ≈ 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈2 kcal/mol, toward denaturant-and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.Fatty acid amide hydrolase (FAAH) 3 is a dimeric, integral membrane protein (1) that brings about the degradation of important fatty acid neurotransmitters, such as oleamide and anandamide (2). These fatty acid amides (FAAs), collectively termed "endocannabinoids," modulate several aspects of mammalian pathophysiology, both at central and peripheral level (3). It is now widely recognized that the biological activity of FAAs depends on the "metabolic control" of their endogenous tone (3), and in the last few years evidence has accumulated that points to FAAH as the key regulator of FAAs concentration in vivo (4, 5). Therefore, this enzyme is becoming the subject of intense investigation, and the design of ad hoc inhibitors able to tune its catalytic activity is a hot spot in medicinal chemistry (for a recent review see Ref. 6). In fact, FAAH inhibitors might represent next-generation therapeutics for the treatment of drug and alcohol abuse (7), anxiety (8), cancer (9), as well as neurodegenerative (10, 11) and vascular (12) diseases. In addition, interest toward FAAH was boosted by the fact that this enzyme is the first and, to date, the only characterized mammalian member of a large group, referred to as the "amidase signature" (AS) family (13). More than 100 members of this class have been reported in the literature, mostly from bacteria or fungi. Despite its quite remarkable size, struct...

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The importance of being dimeric

Cited by 100 publications

References 68 publications

Dimerization Is Responsible for the Structural Stability of Human Sulfotransferase 1A1

Dimerization Is Responsible for the Structural Stability of Human Sulfotransferase 1A1

Specificity versus stability in computational protein design

Closing the Gate to the Active Site

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