Background: Lipoprotein lipase (LPL) clears triglycerides from the blood, and angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. Results: Inhibited LPL is in a complex with ANGPTL4, and upon dissociation LPL regains activity. Conclusion: ANGPTL4 is a reversible, noncompetitive inhibitor of LPL, not an unfolding molecular chaperone as reported previously. Significance: Understanding the mechanism of LPL inhibition supports efforts to develop new therapies for hypertriglyceridemia.

Shelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways.

SUMMARY Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many existing commercial AFMs, making the technique readily available.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure–function properties of these obligate MutSα–MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα–MutLα–DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS–MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.