Zn2+ binding

Fritz G, Heizmann CW (2004) 3D-structures of the Ca2+-and Zn2+-binding SI00 proteins. In Messerschmidt A, Bode W, Cygler M, Handbook of metalloproteins. Wiley, Chichester, pp, 529—540... 

Insights From Endogenous and Engineered Zn2+ Binding Sites in Monoamine Transporters... 

In this chapter, it will be described how we have utilized endogenous and engineered Zn2+ binding sites to explore the structure and molecular function of Na+/Cl -depen-dent neurotransmitter transporters. The work has not only allowed the definition of the first structural constraints in the tertiary structure of this class of transporters, but also provided new insight into both conformational changes accompanying substrate translocation and mechanisms governing conformational isomerization in the translocation cycle. In the chapter, we will also review the theoretical and practical basis for... 

INSIGHTS FROM NATURALLY OCCURRING ZN2+ BINDING SITES IN SOLUBLE PROTEINS... 

The crystal structures of Zn2+ binding proteins not only revealed well-defined tertiary structure constraints to accommodate Zn2+ binding, but also well-defined con-... 

Zn2+ binding sites have been artificially introduced into proteins to stabilize them against denaturation or proteolytic degradation and into enzymes for enhanced regula-... 

Fig. 3. Evidence for an endogenous Zn2+-binding site in the DAT but not in the NET. (A) Zn2+ inhibition of [3H]dopamine uptake in COS-7 cells transiently expressing hDAT (filled circles) or hNET (open circles). (B) Effect of Zn2+ on binding of the cocaine analog [3H]WIN 35,428 to hDAT (filled circles) and hNET (open circles). Values are percent of control ([3H]dopamine uptake or [3H] WIN 35,428 binding in the absence of Zn2+) expressed as means S.E. of 3-5 experiments performed in triplicate.

STRUCTURAL INSIGHT FROM ENGINEERED ZN2+ BINDING SITES IN NA+/CL--DEPENDENT NEUROTRANSMITTER TRANSPORTERS... 

Altogether, the identification of the coordinating residues in the endogenous hDAT Zn2+ binding site followed by the engineering artificial sites have defined an important series of structural constraints in this transporter. This includes not only a series of proximity relationships in the tertiary structure, but also secondary structure relationships. The data also provided information about the orientation of TM7 relative to TM8. A model of the TM7/8 microdomain that incorporates all these structural constraints is shown in Fig. 4 (36). The model is an important example of how structural inferences derived from a series of Zn2+ binding sites can provide sufficient information for at least an initial structural mapping of a selected protein domain. 

Interestingly, we have recently identified a mutation of a tyrosine in the third intracellular loop of the hDAT that causes a major alteration in the conformational equilibrium of the transport cycle, and thus as such is comparable to mutants on G protein-coupled receptors causing constitutive isomerization of the receptor to the active state (66). Most importantly, this conclusion is based on the observation that mutation of the tyrosine completely reverts the effect of Zn2+ at the endogenous Zn2+ binding site in the hDAT (50,51) from potent inhibition of transport to potent stimulation of transport (Fig. 6). In the absence of Zn2+, transport capacity is reduced to less than 1% of that observed for the wild-type, however, the presence of Zn2+ in only micromolar concentrations causes a close to 30-fold increase in uptake (66). Moreover, it is found that the apparent affinities for cocaine and several other inhibitors are substantially decreased, whereas the apparent affinities for substrates are markedly increased (66). Notably, the decrease in apparent cocaine affinity was around 150-fold and thus to date the most dramatic alteration in cocaine affinity reported upon mutation of a single residue in the monoamine transporters (66). 

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