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Table 1 Evaluation of Nef binding to wild-type and HART SH3 domains by surface plasmon resonance (SPR).

From: HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

SH3 domain

ka (M-1s-1) × 105

kd (s-1) × 10-2

KD (μM)

Nef concentrations (μM)

Nef-SF2

Human Hck SH3

1.60 ± 0.06

1.18 ± 0.04

0.07 ± 0.01

0, 0.007, 0.02, 0.062, 0.19, 0.56, 1.67, 5.0

Mouse Hck SH3

5.54 ± 1.77

7.00 ± 0.36

0.13 ± 0.03

0, 0.007, 0.02, 0.062, 0.19, 0.56, 1.67

Mouse Hck HART

14.20 ± 1.40

1.43 ± 0.01

0.01 ± 0.00

0, 0.002, 0.007, 0.02, 0.062, 0.19

Nef-Consensus*

Human Hck SH3

10.00 ± 0.80

11.80 ± 0.20

0.12 ± 0.01

0, 0.02, 0.062, 0.19, 0.56, 1.67, 5.0

Mouse Hck SH3

2.41 ± 0.05

1.85 ± 0.11

0.08 ± 0.01

0, 0.007, 0.02, 0.062, 0.19, 0.56, 1.67

Mouse Hck HART

15.70 ± 0.20

3.24 ± 0.16

0.02 ± 0.00

0, 0.002, 0.007, 0.02, 0.062, 0.19, 0.56

  1. SPR analyses were performed with the recombinant purified Hck SH3 domains covalently attached to the biosensor chip, as described under Materials and Methods. Each untagged, purified Nef protein analyte was serially injected and the Nef-Hck interaction monitored over the range of Nef concentrations shown (SF2 and Consensus variants, as indicated). The kinetics and affinities of binding were determined by fitting the data to a 1:1 Langmuir binding model, with the dissociation constants (KD) and error analysis calculated through the BIAevaluation 4.1 software suite.
  2. *Consensus Nef is based on a sequence alignment of multiple B-clade Nef proteins [40].
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BMC Chemical Biology

ISSN: 1472-6769