Supporting Information for: Antiparallel Coiled Coil Interactions Mediate Homodimerization of the. DNA Damage Repair Protein, PALB2

Transcription

1 Biochemistry Supporting Information for: Antiparallel Coiled Coil Interactions Mediate Homodimerization of the DNA Damage Repair Protein, PALB2 Fei Song,, Minxing Li, Gaohua Liu,, G.V.T. Swapna,, Nourhan S. Daigham,, Bing Xia, Gaetano T. Montelione, *,, and Samuel F. Bunting *, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, USA *To whom correspondence should be addressed: addresses: S 1

2 Supporting Materials and Methods DNA coding for the PALB2 coiled coil domain (residues 1-60, PALB2cc) and BRCA1 coiled coil domain (residues , BRCA1cc) from mouse (Mus musculus) were synthesized (Genscript) and cloned into modified NESG pet21 expression vectors 3, with C- terminal affinity tags (LEHHHHHHSH), yielding the plasmids MmR495A-1-60 and MmR494A These plasmids were then transformed into codon enhanced BL21 (DE3) pmgk E. coli cells, which were then cultured at 37 C in MJ9 minimal medium 4 containing ( 15 NH 4 ) 2 SO 4 and U- 13 C-glucose as the sole sources of nitrogen and carbon. Initial cell growth was carried out at 37 C, and protein expression was induced at 17 C by isopropyl-β-d-thiogalactopyranoside (IPTG). Expressed proteins were purified using an AKTAexpress (GE Healthcare) two-step protocol consisting of HisTrap HP affinity chromatography followed directly by HiLoad 26/60 Superdex 75 gel filtration chromatography, as described elsewhere 3, 5. Sample purity and identity were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, and NMR spectroscopy. Samples of [U- 13 C, 15 N]-PALB2cc and unlabeled BRCA1cc for NMR spectroscopy were concentrated to 1.2 to 1.5 mm in 95% H 2 O / 5% 2 H 2 O solution containing 20 mm MES, 200 mm NaCl, 10 mm DTT, 5 mm CaCl 2 at ph 6.5, except where noted otherwise. All NMR data were collected at 20 C on Bruker AVANCE 600 MHz or 800 MHz NMR spectrometers, processed with NMRPipe 6, and visualized using NMR spectral visualization software SPARKY 7. Complete 1 H, 13 C, and 15 N resonance assignments for PALB2cc were determined using conventional triple resonance NMR methods 8. Stereospecific assignments of Val and Leu isopropyl methyl resonances were determined by the method of Neri et al. 9 using [5% 13 C, U- 15 N]-enriched PALB2cc. Resonance assignments were validated using the Assignment Validation Suite (AVS) software package 10, and deposited in the BioMagResDB (BMRB accession number 27534). 1 H- 15 N heteronuclear NOEs were measured with gradient sensitivity-enhanced 2D heteronuclear NOE approaches 11, 12. The one bond 1 H- 15 N couplings for isotropic and aligned samples of PALB2cc were measured using interleaved [ 15 N- 1 H]-TROSY-HSQC experiments 13. One bond 15 N- 13 C couplings for isotropic and aligned samples of PALB2-CC were measured using NC J- modulation experiments 14. PALB2cc was aligned in a positively charged (50% 3- acrylamidopropyltrimethylammonium chloride + 50% acrylamide) compressed gel 14. The positively charged gel was initially cast in a 3.2 mm diameter plastic tube. The polymerized gel S 2

3 was first washed extensively in deionized water followed by washing with protein buffer to equilibrate the ph. Finally, the gel was washed with deionized water to remove the buffer. The swelled gel (~7 mm diameter) was trimmed to a length of 35 mm and dried in open air for two days. The gel pellet was swollen in a 4 mm Shigemi NMR tube using the protein solution. The plunger of the Shigemi tube was fixed at a height of 12 mm from the bottom of the tube. For the truncated PALB2cc structure determination, initial structure calculations were performed with CYANA , 16, using peak intensities from 3D simultaneous CN-NOESY (τ m = 120 ms), dihedral angle constraints computed by TALOS 17 (φ ± 20 ; ψ ± 20 ), and both N-H N and N-C RDC restraints. The 20 structures with lowest target function out of 100 generated in the final cycle were further were refined by restrained molecular dynamics in explicit water with CNS , 19, using the final NOE derived distance, TALOS dihedral angle, and RDC restraints. Structural statistics and global structure quality factors, including Verify3D 20, ProsaII 21, PROCHECK 22, and MolProbity 23 raw and statistical Z-scores, were computed using the PSVS 1.5 software package 24. The global goodness-of-fit of the final structure ensembles with the NOESY peak list data were determined using the RPF analysis program 25. The final refined ensemble of 20 structures was deposited into the Protein Data Bank (PDB ID, 6e4h). The dissociation constants for the PALB2cc and [L24A]-PALB2 monomer-dimer equilibria, in fast chemical exchange, were calculated from [ 15 N- 1 H]-HSQC NMR chemical shift perturbation data, combining both nitrogen and backbone amide proton chemical shifts and fitting to the following equation for homodimer formation (Eqn 1), as described elsewhere 26. Here, δ D is the chemical shift of the dimer, δ M is the chemical shift of the monomer, δ obs is the observed fast-exchange-averaged chemical shift, K d is the dissociation constant of the homodimer, and c is the total PALB2cc (or [L24A]-PALB2) protein concentration. 15 N and 1 H chemical shifts were combined 26 and used as the single parameters δ. The parameters (δ D -δ M ) and K d were fit simultaneously by grid search to minimize the residual between calculated and observed (δ obs δ 1 ), using an initial estimate of (δ D -δ M ) determined from the chemical shift S 3

4 values of wt-palb2cc or [L24A]-PALB2, respectively, in samples measured at high (i.e. homodimeric) concentrations, and values of δ M determined at dilute sample concentrations. The dissociation constant K d for the slow-exchange equilibrium between wt-palb2cc and the heterodimeric PALB2cc-BRCA1cc complex was determined from the relative intensities of wt-palb2cc [ 15 N- 1 H]-HSQC NMR resonances assigned to homo- and hetero-dimer states, respectively, measured at multiple points in NMR titration experiments using unlabeled BRCA1, and under conditions where wt-palb2cc is primarily homodimeric and unbound BRCA1cc is primarily monomeric. The dissociation constant K d for the fast-exchange monomer-dimer equilibrium between [L24A]-PALB2cc and the heterodimeric [L24A]-PALB2cc : BRCA1cc complex was determined from NMR chemical shift perturbation data for [L24A]-PALB2cc [ 15 N- 1 H]-HSQC NMR resonances upon titration with unlabeled BRCA1cc, using Eqn 2: where K d is the dissociation constant of the L24A]-PALB2cc : BRCA1cc complex, c 1 and c 2 are the total concentrations of [L24A]-PALB2cc and BRCA1cc, respectively, δ 1 is the combined 26 1 H, 15 N chemical shift of 15 N-[L24A]-PALB2cc in the absence of BRCA1cc, and δ 2 is the 1 H, 15 N chemical shift of 15 N-[L24A]-PALB2cc in the [L24A]-PALB2cc : BRCA1cc complex. The parameters (δ 2 - δ 1 ) and K d were fit simultaneously by grid search to minimize the residual between calculated and observed (δ obs - δ 1 ), using an initial estimate of (δ 2 -δ 2 ) determined from the chemical shift values of wt 15 N-PALB2cc bound to unlabeled BRCA1cc at high (i.e. heterodimeric) concentrations, and values of δ 1 determined at dilute concentrations of [L24A]- PALB2 in the absence of BRCA1cc. In all cases, K d values were determined for resonances assigned to several residues, and averaged. S 4

5 Figure S1. Triple resonance NMR connectivity map and summary of other NMR data used to determine resonance assignments and secondary structure for PALB2cc. The first row is the protein sequence of PALB2cc. The next row annotates the secondary structure (helix shown in magenta). Bar graphs of H- N heteronuclear NOE data are shown in blue. Triple resonance intraresidue (i) and 1 15 sequential (s) connectivities for matching intraresidue and sequential C, Cα and Cβ resonances are shown as horizontal red and yellow lines, respectively. The final 11 rows summarize sequential and medium-range NOE data validating the assignments and secondary structure, where dan, dbn, and dnn designate H -H, H -H, and a N b N H -H distances, respectively, between atoms of resonance i and i+1, i+2, i+3, or i+4, the thickness of the N N corresponding connection line is proportional to the corresponding NOE intensity. This image is generated using the AutoAssign software. 1, 2 S 5

6 Figure S2. Assigned [ 1 H- 15 N]-TROSY-HSQC spectrum of homodimeric PALB2cc. The assigned backbone amide resonances are labeled. Data were recorded at 600 MHz. Samples were prepared at ~ 0.2 mm protein concentration in 20 mm MES buffer at ph 6.5, containing 0.02% NaN 3, 10 mm DTT, 5 mm CaCl 2, 200 mm NaCl, 1x Protease Inhibitors, 10% H 2 2O, and 50 μm DSS. S 6

7 Figure S3. Highly-conserved hydrophobic residues Leu17, Leu21, Leu24, Thr31 and Leu35 form an extensive hydrophobic interface. Consurf analysis based on sequence conservation within different species of PALB2 gene are 27 shown on a single chain of PALB2-cc, from the homodimer structure, in space filling representation. Residue coloring ranges from magenta (highly conserved) to cyan (variable), and reflects the degree of residue conservation. The second chain of the dimer is shown in grey line. In forming the coiled-coil dimer, the aliphatic side chains of these key interfacial residues interact with each other in a knobs-into-holes manner. S 7

8 Figure S4. Key long-distance intermolecular NOEs demonstrate unambiguously that the dimer is antiparallel. Selected strips from the 2D X-filtered C13-NOESY spectrum of PALB2cc showing NOEs between the side chain of residue Leu21 in one chain of dimer, with the side chains of Leu32, Thr31 and Tyr28 in another chain of dimer. These residues are located more than 10 Å apart within each α-helical chain of PALB2cc, and thus are too far apart to generate intramolecular NOEs. Rather, they demonstrate the antiparallel orientation of the coiled-coil helices of PALB2cc S 8

9 Figure S5. Multiple Sequence Alignment for N-terminal region of PALB2cc. Residues that are strictly conserved across the organisms used in this alignment are highlighted in dark blue; those which are largely conserved are highlighted in light blue. This image was generated using Clustal Omega software 28 S 9

10 Figure S6. The [ 1 H- 15 N]-HSQC spectrum of [L24A]-PALB2cc confirms that this variant retains some ordered structure. Overlay of 600 MHz [ 1 H- 15 N]-HSQC spectra of wild-type PALB2cc (blue) and [L24A]-PALB2cc (green). The high similarity of these spectra demonstrate that the L24A mutation does not significantly disrupt the 3D structure of PALB2cc. Samples were prepared at ~ 0.2 mm protein concentration in 20 mm MES buffer at ph 6.5, containing 0.02% NaN3, 10 mm DTT, 5 mm CaCl2, 200 mm NaCl, 1x Protease Inhibitors, 10% H2O, and 2 50 μm DSS. S 10

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