Table 2

HIV-1 Nef binding influences the Hck active site.

Condition

Km, ATP (μM)

IC50, NaPP1 (nM)


mHck alone

12.1 ± 2.0

> 1,000

mHck + Nef-SF2

14.8 ± 1.4

> 1,000


mHck-TA alone

62 ± 12.6

53.3 ± 8.2

mHck-TA + Nef-SF2

68 ± 10.7

47.4 ± 6.0

mHck-TA + Nef-Consensus

84 ± 3.5

37.6 ± 4.6


mHck-HART alone

105.2 ± 29.0

n.d.

mHck-HART + Nef-SF2

49.34 ± 5.8

n.d.


mHck-TA-HART alone

181 ± 28.7

8.5 ± 0.7

mHck-TA-HART + Nef-SF2

69 ± 17.6

2.4 ± 0.3

mHck-TA-HART + Nef-Consensus

76 ± 23.1

3.2 ± 0.2


The Km values for ATP and IC50 values for NaPP1 were determined using the Z'Lyte assay (see Materials and Methods). Four forms of purified recombinant mouse Hck (mHck) were used in these experiments: wild-type (mHck); mHck with a modified SH3 domain high affinity RT loop for Nef binding (mHck-HART); mHck with a wild-type SH3 domain and a kinase domain mutation (T338A) that accommodates the inhibitor, NaPP1 (mHck-TA); and a double mutant (mHck-TA-HART). Experiments with Nef contained the recombinant purified viral protein at a 10-fold molar excess, and were performed with two Nef B-clade variants (SF2 and Consensus) in most cases. n.d., not determined.

Pene-Dumitrescu et al. BMC Chemical Biology 2012 12:1   doi:10.1186/1472-6769-12-1

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