. 2004 Mar 18;23(7):1411–1421. doi: 10.1038/sj.emboj.7600114

Table 2.

Ub-binding affinities of different NZF domains

NZF domains^a	NZF sequence^b	K_d (μM)^c
Npl4 (wt)	₅₈₀TSAMWACQHCTFMNQPGTGHCEMCSLPRT	126±26^d
Npl4 (T₁₃L,F14V)	₅₈₀TSAMWACQHCLVMNQPGTGHCEMCSLPRT	>5000^e
Vps36-1 (wt)	₁₇₇VNSENICPACTFANHPQIGNCEICGHRLP	182±19^d
Vps36-1 (T₁₃G)	₁₇₇VNSENICPACGFANHPQIGNCEICGHRLP	1900(100)^e
Vps36-1 (T₁₃G,F₁₄S)	₁₇₇VNSENICPACGSANHPQIGNCEICGHRLP	>5000^e
TAB2 (wt)	₆₆₄EGAQWNCTACTFLNHPALIRCEQCEMPRH	338(1)^f
VPS36-2 (wt)		>5000^e
RanBP2 (wt)	₁₄₇₉KEGQWDCSACLVQNEGSSTKCAACQNPRK	>5000^e
RanBP2 (L13T,V₁₄F)	₁₄₇₉KEGQWDCSACTFQNEGSSTKCAACQNPRK	976(3)^f
RanBP2 (L13T, V₁₄F,A25M)	₁₄₇₉KEGQWDCSACTFQNEGSSTKCAMCQNPRK	92(2)^f
RanBP2 (L13T,V₁₄F,A₂₄E,A25M)	₁₄₇₉KEGQWDCSACTFQNEGSSTKCEMCQNPRK	85±2^g
^aNpl4 (nuclear protein localization gene 4; NCBI NP_542144) forms a heterodimer with Ufd1 that recognizes ubiquitylated target proteins and acts as a cofactor for Cdc48/p97 in the processes of ERAD, RUP, and nuclear envelope closure (Hetzer et al, 2001; Meyer et al, 2002; Tsai et al, 2002). The metazoan Npl4 proteins have a single NZF domain, which is also found in some but not all fungal Npl4 proteins.
Vps36 (vacuolar protein sorting protein 36; NCBI NP_013521) is a component of the yeast ESCRT-II complex and is required for the sorting of ubiquitylated protein cargos into the multivesicular body (Babst et al, 2002; Katzmann et al, 2002). Yeast Vps36 contains two NZF domains, one of which binds Ub (Vps36-1) and the other which does not (Vps36-2). Note that these domains are not conserved in the mammalian EAP45/Vps36 protein.
TAB2 (TAK1-binding protein 2; NCBI NP_055908) is a subunit of the heterotrimeric TRIKA2 kinase complex, a Ub-dependent MKK and IKK kinase involved in IL-1 receptor signaling (Wang et al, 2001). The mammalian and amphibian proteins have a single NZF domain.
RanBP2 (Ran-binding protein 2/Nup358; NCBI NM_006267) is a Ran-GDP-binding subunit of the nuclear pore complex (Nakielny et al, 1999; Yaseen and Blobel, 1999). This NZF domain corresponds to the third of eight NZF domains in human RanBP2.
^bSubscripts denote the starting position of each NZF domain within the relevant protein. Underlined residues coordinate zinc, and residues mutated from the wt sequence are shown in bold.
^cK_d values were determined in BIACORE biosensor measurements of soluble Ub binding to immobilized GST-NZF fusion proteins at 20°C in a buffer of 25 mM Tris–HCl pH 7.0, 2 μM ZnCl₂, 0.005% P20, 50 μg/ml bovine serum albumin, and 2 mM DTT. Note that several of these measurements were reported previously (Wang et al, 2003), but are also included here for completeness.
^dDissociation constant is the mean of four or more independent measurements and the error is the standard deviation in the measurements.
^eExtrapolated from less than 50% saturation binding at 1.5 mM free Ub protein concentration.
^fDissociation constant and error were estimated from a statistical fit of a single binding isotherm derived from triplicate measurements at seven different Ub concentrations (2.1–1500 μM). Values in parentheses are statistical errors estimated from the fit of the data to a simple 1:1 binding model.
^gDissociation constant is the mean of two independent measurements and the error is the range in the two measurements.