MRF Project NE-1028 (to J. M. B.), National Institute of Allergy and Infectious Diseases (NIAID) grant 1R01AI139100-01 (to J. M. B.) and as a Community Outreach Project of the National Institutes of Health NIGMS Protein Structure Initiative Grant U54 GM094597 (to G. T. M. and T. S.). Support was also provide by NIGMS Grants R01 GM120574 (to G. T. M.) and S10 OD018207 (to G. T. M.). G. T. M. is a founder of Nexomics Biosciences, Inc. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S19, Tables S1-S4, and supporting Refs. 1-18. The atomic coordinates and structure factors (code 2KY9) have been deposited in the Protein Data Bank (http://wwpdb.org/). The NMR chemical shift data of this paper are available from the Biological Magnetic Resonance Data Bank under BMRB accession numbers 16942 and 27741.

Deficits in DNA damage repair pathways are the root cause of several human cancers. In mammalian cells, DNA double-strand break repair is carried out by multiple mechanisms, including homologous recombination (HR). PALB2 (Partner and Localizer of BRCA2), which is an essential factor for HR, binds to the breast cancer susceptibility 1 (BRCA1) protein at DNA double-strand breaks. At the break site, PALB2 also associates with breast cancer susceptibility 2 (BRCA2) protein, to form a multi-protein complex that facilitates HR. The BRCA1-PALB2 interaction is mediated by association of predicted helical coiled-coil regions in both proteins. PALB2 can also homodimerize through the formation of a coiled coil, by self-association of helical elements at the N-terminus of the PALB2 protein, and this homodimerization has been proposed to regulate the efficiency of HR. We have produced a segment of PALB2 (PALB2cc), which forms α-helical structures, and which assembles into stable homodimers or heterodimers with a PALB2-interacting segment of BRCA1 (BRCA1cc). The three-dimensional structure of the homodimer formed by PALB2cc was determined by solution NMR spectroscopy. This PALB2cc homodimer is a classical anti-parallel coiled-coil leucine zipper. NMR chemical shift perturbation studies were used to study dimer formation for both the PALB2cc homodimer and the PALB2cc:BRCA1cc heterodimer. Mutation of residue Leu24 of PALB2cc significantly reduces its homodimer stability, but has a more modest effect on the stability of the heterodimer formed between PALB2cc and BRCA1cc. We show that mutation of Leu24 leads to genomic instability and reduced cell viability after treatment with agents that induce DNA double-strand breaks. These studies may allow the identification of distinct mutations of PALB2 which selectively disrupt homodimeric versus heterodimeric interactions, and reveal the specific role of PALB2 homodimerization in HR.

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the three primary experimental means of characterizing macromolecular structures, including protein structures. Structure determination by solution NMR spectroscopy has traditionally relied heavily on distance restraints derived from nuclear Overhauser effect (NOE) measurements. While structure determination of proteins from NOE-based restraints is well understood and broadly used, structure determination from Residual Dipolar Couplings (RDCs) is relatively less well developed. Here, we describe the new features of the protein structure modeling program REDCRAFT and focus on the new Adaptive Decimation (AD) feature. The AD plays a critical role in improving the robustness of REDCRAFT to missing or noisy data, while allowing structure determination of larger proteins from less data. In this report we demonstrate the successful application of REDCRAFT in structure determination of proteins ranging in size from 50 to 145 residues using experimentally collected data, and of larger proteins (145 to 573 residues) using simulated RDC data. In both cases, REDCRAFT uses only RDC data that can be collected from perdeuterated proteins. Finally, we compare the accuracy of structure determination from RDCs alone with traditional NOE-based methods for the structurally novel PF.2048.1 protein. The RDC-based structure of PF.2048.1 exhibited 1.0 Å BB-RMSD with respect to a high-quality NOE-based structure. Although optimal strategies would include using RDC data together with chemical shift, NOE, and other NMR data, these studies provide proof-of-principle for robust structure determination of largely-perdeuterated proteins from RDC data alone using REDCRAFT.

AbstractNuclear Magnetic Resonance (NMR) spectroscopy is one of the two primary experimental means of characterizing macromolecular structures, including protein structures. Structure determination by NMR spectroscopy has traditionally relied heavily on distance restraints derived from nuclear Overhauser effect (NOE) measurements. While structure determination of proteins from NOE-based restraints is well understood and broadly used, structure determination by NOEs imposes increasing quantity of data for analysis, increased cost of structure determination and is less available in the study of perdeuterated proteins. In the recent decade, Residual Dipolar Couplings (RDCs) have been investigated as an alternative source of data for structural elucidation of proteins by NMR. Several methods have been reported that utilize RDCs in addition to NOEs, and a few utilize RDC data alone. While these methods have individually demonstrated some successes, none of these methods have exposed the full potential of protein structure determination from RDCs. To date, structure determination of proteins from RDCs is limited to small proteins (less than 8.5 kDa) using RDC data from many alignment media (>3) that cannot be collected from larger proteins. Here we present the latest version of the REDCRAFT software package designed for structure determination of proteins from RDC data alone. We have demonstrated the success of REDCRAFT in structure determination of proteins ranging in size from 50 to 145 residues using experimentally collected data and large proteins (145 to 573 residues) using simulated RDC data that can be collected from perdeuterated proteins. Finally, we demonstrate the accuracy of structure determination of REDCRAFT from RDCs alone in application to the structurally novel PF.2048 protein. The RDC-based structure of PF.2048 exhibited 1.0 Å of BB-RMSD with respect to the NOE-based structure by only using a small amount of backbone RDCs (∼3 restraints per residue) compared to what is required by other approaches.Author SummaryResidual Dipolar Couplings have the potential to reduce the cost and the time needed to characterize protein structures. In addition, RDC data have been demonstrated to concurrently elucidate structure of proteins, perform assignment of resonances, and be used in characterization of the internal dynamics of proteins. Given all the advantages associated with the study of proteins from RDC data, based on the statistics provided by the Protein Databank (PDB), surprisingly the only 124 proteins (out of nearly 150,000 proteins) have utilized RDCs as part of their structure determination. Even a smaller subset of these proteins (approximately 7) have utilized RDCs as the primary source of data for structure determination. The impeding factor in the use of RDCs is the challenging computational and analytical aspects of this source of data. In this report, we demonstrate the success of the REDCRAFT software package in structure determination of proteins using RDC data that can be collected from small and large proteins in a routine fashion. REDCRAFT accomplishes the challenging task of structure determination from RDCs by introducing a unique search and optimization technique that is both robust and computationally tractable. Structure determination from routinely collectable RDC data using REDCRAFT can lead to faster and cheaper study of larger and more complex proteins by NMR spectroscopy in solution state.

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