Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding Jamie A. Moroco, John Jeff Alvarado, Ryan P. Staudt, Haibin Shi, Thomas E. Wales, Thomas E. Smithgall and John R. Engen

SUPPLEMENTARY FIGURE LEGENDS

Figure S1. Coverage Maps. Peptic peptides identified and monitored during HX MS experiments for (a) Nef core and (b) Hck SH3-SH2. Each blue bar under the sequence indicates a peptide. Minimum peptide coverage was 87% for the Nef core and 73% for the Hck SH3-SH2 protein. The green arrow in (b) indicates the start of the Hck SH3 protein construct. While C-terminal 6x-histidine tags were present in each protein, they are hidden here as they were not followed in the experiments. The peptides shown here were coincident throughout all experiments in this work but individual comparison experiments yielded additional peptides (not shown) which were included in the uptake plots (Supplementary Figs. S2, S6, S7, S9, S11, S12). Figure S2. Deuterium uptake plots comparing full-length Nef to Nef core. Deuterium uptake plots comparing measured relative deuterium levels in full-length (FL) Nef (red circles) and Nef core (black circles). The residue numbering of each peptide is shown in the top left of each graph. Because Nef SF2 contains a 4 residue insertion, the numbering begins at negative two to maintain Nef NL43 numbering in the core. Plots with negative residue numbering are indicated with a superscript (a). The y-axis maximum of each plot is the theoretical amount of deuterium that could be exchanged into backbone amide positions of each peptide. Because the Nef core begins at residue 58, peptides between residues 1-57 are only present in the FL-Nef construct. Yellow stars represent the non-overlapping peptides used to generate the linear difference map shown in main Figure 2. Figure S3. Kinetics of Nef protein interactions with Hck SH3 and SH3-SH2 domains. Protein-protein interactions were measured in real time by surface plasmon resonance (SPR). Recombinant Hck SH3 and SH3-SH2 proteins were immobilized covalently as ligand on carboxymethyl dextran SPR chips (Reichert) using standard EDC/NHS amine coupling chemistry. Nef proteins were then injected in triplicate over a range of concentrations (0.04 to 3.3 μM) at a flow rate of 30 μL/min for one min until equilibrium was reached, followed by a 3 min dissociation phase. Kinetic rate constants were calculated from reference-corrected sensorgrams using TraceDrawer software, and were best fit by 1:1 Langmuir (SH3) or two-state (SH3-SH2) binding models. Equilibrium dissociation constants (KD values) calculated from the resulting rate constants are shown, ± S.D. N/A, not applicable. Representative sensorgrams from each experiment are shown below the Table. The SPR responses recorded at each concentration are shown as colored points, with the fitted curves superimposed as black lines.

1

  

Figure S4. Analytical size-exclusion chromatography of Nef:Hck complexes. Part (a) shows size-exclusion chromatography (SEC) elution profiles of the purified Nef core:Hck SH3 complex, the Nef-D123N:Hck SH3 complex, and individual proteins making up the complexes. Part (b) shows SEC elution profiles of the purified Nef core:Hck SH3-SH2 complex, the Nef-D123N:Hck SH3-SH2 complex, and individual proteins making up the complexes. The positions of the elution peaks for the protein standards bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (12.5 kDa) and aprotinin (6.5 kDa) are indicated with grey dotted lines. The positions of the Nef core complexes with SH3 and SH3-SH2 are also indicated on the plots for reference (green dotted lines). Note that the stoichiometry of an identical Nef core complex with the Hck SH3:SH2 protein was previously determined by SEC combined with multi-angle light scattering to be a dimer of complexes [1]. Figure S5. The Hck SH3 domain is protected upon Nef core binding. Comparison of HX into Hck SH3, Hck SH3-SH2, and complexes of SH3:Nef core and SH3-SH2:Nef core. [NOTE: in describing HX MS data for complexes, the first protein in the X:Y complex nomenclature is the one for which HX MS was monitored, e.g. SH3:Nef core follows exchange in SH3, etc.] Both (a) vertical difference maps and (b) selected deuterium incorporation graphs are shown. All deuterium incorporation graphs, are shown in Supplementary Fig. S6. The deuterium level in the free form (Hck SH3 or Hck SH3-SH2) was subtracted from the deuterium level in the complex (Hck SH3-SH2:Nef, or Hck SH3:Nef) and the differences color-coded as indicated in the legend. The differences found in non-overlapping peptides (yellow stars in Supplementary Fig. S6), displayed vertically from N- to C-terminus, are indicated for the corresponding exchange times (shown horizontally). Differences are shown as more intense red (increased deuterium incorporation) or blue (decreased deuterium incorporation). Deuterium incorporation graphs for selected peptides (b) highlight regions where clear differences were seen in SH3 (peptides i, ii, iii, iv, left side) and in SH2 (peptides v, vi, vii and viii, right side). Figure S6. Deuterium uptake plots comparing Hck SH3 and Hck SH3-SH2 alone and in complex with Nef core. Deuterium uptake plots comparing measured relative deuterium levels in Hck SH3 alone (gray circles), Hck SH3:Nef core complex (blue circles), Hck SH3-SH2 alone (black circles), and Hck SH3-SH2:Nef core complex (red circles). The residue numbering of each peptide is shown at the top left of each graph. The y-axis maximum of each plot is the theoretical amount of deuterium that could be exchanged into backbone amide positions of each peptide. Peptides between residues 80-143 are present in all states shown, while residues 144-245 are only present in states containing Hck SH3-SH2. Yellow stars represent the non-overlapping peptides used to generate the linear difference map shown in Supplementary Fig. S5a.

2

  

Figure S7. Deuterium uptake plots comparing the Nef core alone and in complex with the Hck SH3 or Hck SH3-SH2 domains. Deuterium uptake plots comparing measured relative deuterium levels in the Nef core (black circles), Nef core:Hck SH3 (blue circles), and Nef core:Hck SH3-SH2 (red circles). The residue numbering of each peptide is shown at the top left of each graph. The y-axis maximum of each plot is the theoretical amount of deuterium that could be exchanged into backbone amide positions of each peptide. Yellow stars represent the non-overlapping peptides used to generate the linear difference map shown in the main text, Figure 3. Figure S8. D123N mutation has little effect on Nef core structure. (a) Comparison of deuterium uptake by the wild-type versus D123N mutant Nef core proteins is presented in a vertical difference map of the Nef core secondary structure. The deuterium level of each peptide from wild-type Nef core was subtracted from that in Nef-D123N and the differences are color-coded as indicated in the legend. The differences in non-overlapping peptides (yellow stars in Supplementary Fig. S9) are displayed vertically from N- to C-terminus for each exchange time point (10 s to 4 h; shown horizontally). Protection in wild-type Nef core is represented as shades of blue and exposure in shades of red. The location of the D123N mutation is indicated in orange on the difference maps and the structures in panel (c). (b) Deuterium incorporation graphs for selected peptides i, ii, iii, iv (the same peptides highlighted in Figs. 2 – 4 in the main text), and v, the peptide containing the D123N mutation. Uptake curves from wild-type Nef core peptides are shown in black and Nef-D123N peptides are in red. (c) The differences observed in wild-type Nef core after 10 s (left) or 4 h (right) of deuterium exchange are plotted onto a monomer of Nef from the 1EFN Nef:SH3 dimer structure. All observed differences are minimal and localized to the region surrounding the mutation. Deuterium incorporation graphs for all peptic peptides from this experiment are shown in Supplementary Fig. S9. Figure S9. Deuterium uptake plots comparing wild-type Nef core and Nef-D123N. Deuterium uptake plots comparing wild-type Nef core (black circles) to Nef-D123N (red circles). The residue numbering of each peptide is shown at the top left of each graph. The y-axis maximum of each plot is the theoretical amount of deuterium that could be exchanged into backbone amide positions of each peptide. Peptides containing the D123N mutation are indicated with asterisks (*). Yellow stars represent the non-overlapping peptides used to generate the linear difference map shown in Supplementary Fig. S8a. Figure S10. Hck SH3 domain is protected upon Nef-D123N binding. A comparison of HX into Hck SH3 and Hck SH3-SH2 upon binding to the Nef-D123N mutant in (a) vertical difference maps and (b) deuterium incorporation graphs for selected peptides. All deuterium incorporation graphs are shown in Supplementary Fig. S11. The deuterium level in the free form (Hck SH3 or Hck SH3-SH2) was subtracted from the deuterium level in the complex (Hck SH3-SH2:Nef-D123N, or Hck SH3:Nef-D123N) and the differences color-coded as indicated in the legend. The differences found in non-overlapping peptides (yellow stars in Supplementary Fig. S11), displayed vertically from N- to C-terminus, are indicated for the corresponding exchange times (shown horizontally). Greater differences are shown as more intense red (increased deuterium incorporation) or blue (decreased deuterium incorporation). Deuterium incorporation graphs (Hck SH3 is shown in gray, Hck SH3:Nef-D123N in blue, Hck SH3-SH2 in black, and Hck SH3-SH2:Nef-D123N in red ) for

3

  

selected peptides (b) highlight regions where clear differences were seen in SH3 (peptides i, ii, iii, iv, left side) and in SH2 (peptides v, vi, vii and viii, right side). Figure S11. Deuterium uptake plots comparing Hck SH3 and Hck SH3-SH2 alone and in complexes with Nef-D123N. Deuterium uptake plots comparing measured relative deuterium levels in Hck SH3 alone (gray circles), Hck SH3-SH2 alone (black circles), Hck SH3:Nef-D123N complex (blue circles), and Hck SH3-SH2:Nef-D123N complex (red circles). The residue numbering of each peptide is shown at the top left of each graph. The y-axis maximum of each plot is the theoretical amount of deuterium that could be exchanged into backbone amide positions of each peptide. Peptides between residues 80-143 are present in all of the states shown, while 144-245 are only present in states containing Hck SH3-SH2. Yellow stars represent the non-overlapping peptides used to generate the linear difference map shown in Supplementary Fig. S10. Figure S12. Deuterium uptake plots comparing Nef-D123N alone and in complex with Hck SH3 or Hck SH3-SH2. Deuterium uptake plots comparing measured relative deuterium levels in Nef-D123N (black circles), Nef-D123N:Hck SH3 (blue circles), and Nef-D123N:Hck SH3-SH2 (red circles). The residue numbering of each peptide is shown in the top left of each graph. The y-axis maximum of each plot is the theoretical amount of deuterium that could be exchanged into backbone amide positions of each peptide. Yellow stars represent the non-overlapping peptides used to generate the linear difference map shown in the main text, Figure 4. Peptides containing the Nef-D123N mutation are indicated with asterisks (*). SUPPLEMENTARY REFERENCES [1] J.J. Alvarado, S. Tarafdar, J.I. Yeh, T.E. Smithgall, Interaction with the Src homology (SH3-SH2)

region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment, J Biol Chem 289 (2014) 28539-28553.

4

Figure S1

(a) HIV Nef

(b) Hck SH3-SH2

5

Deuterium Uptake: FL-Nef and Nef core

Figure S2

a a

FL-NefNef coreLinear map

a Negative numbering6

Figure S3

Hck Ligand Nef Analyte ka1, 1/M · s kd1, 1/s ka2, 1/M · s kd2, 1/s KD, M

SH3 Full-length 2.84 x 105 4.97 x 10-2 N/A N/A1.80 ± 0.42 x

10-7

SH3 wild-type core 2.24 x 105 2.76 x 10-2 N/A N/A1.28 ± 0.31 x

10-7

SH3 D123N core 2.90 x 105 3.00 x 10-2 N/A N/A1.19 ± 0.44 x

10-7

SH3-SH2 Full-length 1.33 x 105 5.34 x 10-2 3.44 x 10-3 3.29 x 10-3 4.29 ± 1.32 x 10-7

SH3-SH2 wild-type core 6.29 x 104 5.65 x 10-2 1.06 x 10-2 4.72 x 10-3 4.08 ± 0.45 x 10-7

SH3-SH2 D123N core 5.72 x 104 4.93 x 10-2 9.03 x 10-3 4.88 x 10-3 4.75 ± 0.99x 10-7

7

8 10 12 14 16 18

0.0

0.2

0.4

0.6

0.8

1.0

Retention Volume (mL)

Abs

orb a

n ce

(mA

U)

66 kDa 29 kDa 12.5 kDa 6.5 kDa

Nef core:Hck SH3

Nef-D123N:Hck SH3

Hck SH3

Nef-D123NNef core

MW standardsNef core:Hck SH3-SH2

Hck SH3 and Nef complexes

8 10 12 14 16 18

0

1

2

3

4

5

Retention Volume (mL)

Abs

orb

an

ce

(mA

U)

66 kDa 29 kDa 12.5 kDa 6.5 kDa

Nef core:Hck SH3-SH2

Nef-D123N:Hck SH3-SH2

Hck SH3-SH2

Nef-D123NNef core

MW standardsNef core:Hck SH3

Hck SH3-SH2 and Nef complexes

(a)

(b)

Figure S4

8

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐1.5895 ‐1.0288 ‐1.1338 ‐0.5826 ‐0.5069

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

‐0.2017 ‐0.2024 ‐0.7612 ‐1.3534 ‐0.6336

100 100 100 100 100

100 100 100 100 100

0.0152 0.2018 0.0049 0.1402 0.096

0.0152 0.2018 0.0049 0.1402 0.096

0.0152 0.2018 0.0049 0.1402 0.096

0.0152 0.2018 0.0049 0.1402 0.096

0.0152 0.2018 0.0049 0.1402 0.096

0.0152 0.2018 0.0049 0.1402 0.096

0.0152 0.2018 0.0049 0.1402 0.096

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.3263 ‐0.051 ‐0.8117 ‐1.6287 ‐0.8684

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.8178 ‐0.6965 ‐1.868 ‐1.93 ‐0.9361

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.1372 0.0404 ‐0.1264 ‐0.0432 0.012

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

‐0.0161 ‐0.0592 0.1501 0.1739 0.0329

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐1.3612 ‐1.2687 ‐1.2774 ‐0.6692 ‐0.5424

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

‐0.1392 ‐0.3816 ‐0.9594 ‐1.1656 ‐0.5339

100 100 100 100 100

100 100 100 100 100

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.1902 ‐0.0974 ‐0.0916 0.0147 ‐0.0705

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.3145 ‐0.3907 ‐1.1012 ‐1.4986 ‐0.7234

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.655 ‐0.8266 ‐1.9504 ‐1.6369 ‐0.6664

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

‐0.1536 ‐0.122 ‐0.2088 ‐0.0909 ‐0.1018

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.3369 ‐0.1569 ‐0.0446 0.0143 ‐0.171

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

‐0.1541 ‐0.0419 0.295 0.7256 0.1534

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.5174 ‐0.1764 0.1778 0.342 ‐0.1846

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

‐0.4079 ‐0.1915 0.731 1.7015 0.4226

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

‐0.2081 ‐0.1206 0.0991 0.1568 ‐0.1092

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

‐0.1766 ‐0.1576 0.1838 0.5735 0.7245

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

0.0477 0.2332 0.185 0.0945 ‐0.0616

i. 90-101

SH3SH3:Nef

SH3-SH2:NefSH3-SH2

v.159-168

ii. 102-108 vi. 173-185

iii. 118-125 vii. 186-205

iv. 126-135 viii. 223-236

SH3:Nef

7580

100

200

245

SH3-SH2:NefN

C

RT

loo

p

iii

ii

i

iv

vii

vi

v

viii

10s 1m 10m 1h 4h

143150

Rel

ativ

e U

ptak

e (D

a)

Exposure Time (min)

100

‐3 ‐1.51 ‐1.49 ‐0.99 0 0.51 1.01 1.51 30.5 1.0 1.5 2.0-2.0 -1.5 -1.0 -0.5

No Coverage

Protection Exposure0

Dcomplex – DHckSH3/SH3-SH2 (Da)

(a) (b)

Residue #

Figure S5

9

Deuterium Uptake: Hck SH3/SH3-SH2 alone and in complex with Nef core

SH3:Nef

SH3-SH2:Nef

SH3

SH3-SH2

Linear map

Figure S6

10

Nef:SH3

Nef:SH3-SH2

Nef Core

Linear map

Figure S7

Deuterium Uptake: Nef core alone and in complex with Hck SH3/SH3-SH2

11

Figure S8

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

‐0.161 ‐0.0747 ‐0.048 ‐0.0193 0.0215

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.2893 ‐0.1285 ‐0.2441 ‐0.1806 ‐0.051

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

‐0.0432 ‐0.134 ‐0.2037 ‐0.6606 ‐0.8226

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.042 ‐0.1395 ‐0.6265 ‐0.5412 ‐0.4609

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

0.8214 0.4346 0.1535 0.1547 0.1086

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.1306 ‐0.0685 ‐0.0002 ‐0.0679 ‐0.0102

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.2426 ‐0.1121 ‐0.1061 ‐0.1031 0.1135

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.1922 ‐0.0754 ‐0.075 ‐0.1448 0.0447

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.0999 ‐0.0677 ‐0.0349 ‐0.1592 0.018

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

‐0.3116 ‐0.0763 ‐0.0279 ‐0.116 0.033

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

Exposure Time (min)

(a) (b)

Nef monomerPDB: 1EFN

(c)

Exposure in D123N at 10s

Protection in D123N at 4h

Residue #

10s 1m 10m 1h 4h

i

ii

iii

iv

150

100

200

58

205

D123N

Rel

ativ

e U

pta

ke (

Da)

Nef Core WTNef-D123N

N

C

A

B

C

D

Pxx

P

A

B

C

D

‐3 ‐1.51 ‐1.49 ‐0.99 0 0.51 1.01 1.51 30.5 1.0 1.5 2.0-2.0 -1.5 -1.0 -0.5

Protection Exposure0

DNef-D123N – DNefWT (Da)

No Coverage100

v

i. 67-80

ii. 86-100

iv. 113-121

v. 122-139

iii. 100-112

A B

D123N

12

Peptide contains mutation

Deuterium Uptake: Nef core WT and Nef-D123N

Nef-D123N

Nef core

*Linear map

*

*****

Figure S9

13

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

‐0.0491 ‐0.0044 0.0888 0.0839 0.1215

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐1.6759 ‐1.6859 ‐1.497 ‐0.9588 ‐0.7442

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

‐0.0676 ‐0.2906 ‐1.0859 ‐1.5905 ‐1.1003

100 100 100 100 100

100 100 100 100 100

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2458 ‐0.1607 ‐0.1441 ‐0.1679 ‐0.1842

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.2594 ‐0.2548 ‐1.182 ‐2.0387 ‐1.482

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.8604 ‐1.1497 ‐2.5899 ‐2.8345 ‐1.7772

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

‐0.0974 ‐0.0487 ‐0.1364 ‐0.1791 ‐0.019

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

‐0.331 ‐0.1447 0.2272 ‐0.1361 ‐0.0583

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

0.0501 0.1401 1.2747 0.809 0.1412

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1594 ‐0.0808 0.5151 0.2094 ‐0.0487

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

‐0.1535 0.1432 2.3827 1.5399 0.3006

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

‐0.0536 0.0634 1.227 1.5468 1.3271

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

0.3352 0.3064 0.1483 ‐0.0941 ‐0.072

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐1.4872 ‐1.6603 ‐1.2486 ‐0.8545 ‐0.8926

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

‐0.0272 ‐0.3017 ‐0.6742 ‐1.5716 ‐1.2078

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.2491 ‐0.1959 ‐0.7055 ‐1.9288 ‐1.7274

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.7991 ‐1.0927 ‐2.1288 ‐3.0035 ‐2.3444

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

‐0.123 0.0045 ‐0.0586 ‐0.161 ‐0.0631

SH3:Nef-D123N

7580

100

200

245

SH3-SH2:Nef-D123NN

C

RT

loo

p

10s 1m 10m 1h 4h

143150

Rel

ativ

e U

ptak

e (D

a)

Exposure Time (min)

iii

ii

i

iv

vii

vi

v

viii

100

‐3 ‐1.51 ‐1.49 ‐0.99 0 0.51 1.01 1.51 30.5 1.0 1.5 2.0-2.0 -1.5 -1.0 -0.5

No Coverage

Protection Exposure0

Dcomplex – DHckSH3/SH3-SH2 (Da)

ii. 102-108 vi. 173-185

iii. 118-125 vii. 186-205

iv. 126-135 viii. 223-236

i. 90-101 v.159-168

SH3SH3:Nef-D123N

SH3-SH2:Nef-D123NSH3-SH2

Residue #

(a) (b)

Figure S10

14

SH3:Nef-D123N

SH3-SH2:Nef-D123N

SH3

SH3-SH2

Linear map

Figure S11

Deuterium Uptake: Hck SH3/SH3-SH2 alone and in complex with Nef-D123N

15

Nef-D123N:SH3

Nef-D123N:SH3-SH2

Nef-D123N

Linear map

Peptide contains mutation*

*

* ** * *

Figure S12

Deuterium Uptake: Nef-D123N alone and in complex with Hck SH3/SH3-SH2

16