Coiled-coil sequences in proteins consist of heptad repeats containing two characteristic hydrophobic positions. The role of these buried hydrophobic residues in determining the structures of coiled coils was investigated by studying mutants of the GCN4 leucine zipper. When sets of buried residues were altered, two-, three-, and four-helix structures were formed. The x-ray crystal structure of the tetramer revealed a parallel, four-stranded coiled coil. In the tetramer conformation, the local packing geometry of the two hydrophobic positions in the heptad repeat is reversed relative to that in the dimer. These studies demonstrate that conserved, buried residues in the GCN4 leucine zipper direct dimer formation. In contrast to proposals that the pattern of hydrophobic and polar amino acids in a protein sequence is sufficient to determine three-dimensional structure, the shapes of buried side chains in coiled coils are essential determinants of the global fold.

Lung alveoli are lined by squamous alveolar epithelial type 1 (AT1) epithelial cells that facilitate gas exchange, and neighboring AT2 cells that synthesize and secrete surfactant. Alveoli are maintained by intermittent activation of rare ‘bifunctional’ AT2 cells that retain surfactant biosynthesis function but also serve as stem cells, generating new AT1 cells and self-renewing throughout adult life. While stem cell proliferation is controlled by EGFR/KRAS signaling, how the stem cells are selected, maintained, and the fates of their daughter cells controlled are unknown. Here we show that expression of the Wnt target gene Axin2 in mouse lung identifies a rare, stable subpopulation of AT2 cells with stem cell activity. Many lie near single fibroblasts that express Wnt5a and other Wnt genes, and genetically targeting Wnt secretion by fibroblasts depletes the Axin2+ AT2 stem cell population. Axin2 turns off when daughter cells leave the Wnt niche and transdifferentiate into AT1 cells, and sustaining Wnt signaling blocks transdifferentiation whereas abrogation of Wnt signaling promotes it, both in vivo and in vitro. Upon severe alveolar epithelial injury, Axin2 is induced throughout the AT2 population, recruiting ‘ancillary’ AT2 cells into a progenitor role. Niche expression of Wnt5a and the Wnt secretion mediator Porcupine is unchanged by injury, but Wnt7b and several other Wnt genes are broadly induced along with Porcupine in AT2 cells, and pharmacologic or genetic inhibition of this autocrine Wnt signaling impairs the AT2 proliferative response. The results support a model in which individual AT2 cells reside in single cell fibroblast niches that provide a short-range paracrine (or "juxtacrine") Wnt signal that selects and maintains alveolar stem cell identity and proliferative capacity, while severe injury induces AT2 autocrine Wnt signals that transiently expand the stem cell pool during repair.

Subunit oligomerization in many proteins is mediated by short coiled-coil motifs. These motifs share a characteristic seven-amino-acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Despite this common pattern, different sequences form two-, three- and four-stranded helical ropes. We have investigated the basis for oligomer choice by characterizing variants of the GCN4 leucine-zipper dimerization domain that adopt trimeric or tetrameric structures in response to mutations at the a and d positions. We now report the high-resolution X-ray crystal structure of an isoleucine-containing mutant that folds into a parallel three-stranded, alpha-helical coiled coil. In contrast to the dimer and tetramer structures, the interior packing of the trimer can accommodate beta-branched residues in the most preferred rotamer at both hydrophobic positions. Compatibility of the shape of the core amino acids with the distinct packing spaces in the two-, three- and four-stranded conformations appears to determine the oligomerization state of the GCN4 leucine-zipper variants.

Recent advances in computational techniques have allowed the design of precise side-chain packing in proteins with predetermined, naturally occurring backbone structures. Because these methods do not model protein main-chain flexibility, they lack the breadth to explore novel backbone conformations. Here the de novo design of a family of alpha-helical bundle proteins with a right-handed superhelical twist is described. In the design, the overall protein fold was specified by hydrophobic-polar residue patterning, whereas the bundle oligomerization state, detailed main-chain conformation, and interior side-chain rotamers were engineered by computational enumerations of packing in alternate backbone structures. Main-chain flexibility was incorporated through an algebraic parameterization of the backbone. The designed peptides form alpha-helical dimers, trimers, and tetramers in accord with the design goals. The crystal structure of the tetramer matches the designed structure in atomic detail.

DNA is thought to behave as a stiff elastic rod with respect to the ubiquitous mechanical deformations inherent to its biology. Here, we measure the mean and variance of end-to-end length for a series of DNA double helices in solution, using small-angle X-ray scattering interference between gold nanocrystal labels. The data rule out the conventional elastic rod model. Specifically, the variance in end-to-end length follows a quadratic dependence on the number of base pairs rather than the expected linear dependence. Absent applied tension, DNA is at least one order of magnitude softer than measured by single-molecule stretching experiments. Our observations indicate that DNA stretching is cooperative over more than two turns of the DNA double helix, and support the idea of long-range allosteric communication through DNA structure.

Specific protein-protein interactions are crucial in signaling networks and for the assembly of multi-protein complexes, and represent a challenging goal for protein design. Optimizing interaction specificity requires both positive design, the stabilization of a desired interaction, and negative design, the destabilization of undesired interactions. Currently, no automated protein-design algorithms use explicit negative design to guide a sequence search. We describe a multi-state framework for engineering specificity that selects sequences maximizing the transfer free energy of a protein from a target conformation to a set of undesired competitor conformations. To test the multi-state framework, we engineered coiled-coil interfaces that direct the formation of either homodimers or heterodimers. The algorithm identified three specificity motifs that have not been observed in naturally occurring coiled coils. In all cases, experimental results confirm the predicted specificities.

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The human body at cellular resolution: the NIH Human Biomolecular Atlas Program HuBMAP consortium* Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at singlecell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions. t he human body is an incredible machine. Trillions of cells, organized across an array of spatial scales and a multitude of functional states, contribute to a symphony of physiology. While we broadly know how cells are organized in most tissues, a comprehensive understanding of the cellular and molecular states and interactive networks resident in the tissues and organs, from organizational and functional perspectives, is lacking. The specific three-dimensional organization of different cell types, together with the effects of cell-cell and cell-matrix interactions in their natural milieu, have a profound impact on normal function, natural ageing, tissue remodelling, and disease progression in different tissues and organs. Recently, new technologies have enabled the molecular characterization of a multitude of cell types 1-4 and mapping of their spatial relationships in complex tissues at unprecedented scale and single-cell resolution. These advances create the opportunity to build a high-resolution atlas of three-dimensional maps of human tissues and organs. HuBMAP (https://commonfund.nih.gov/hubmap) is an NIHsponsored program with the goals of developing an open framework and technologies for mapping the human body at cellular resolution as well as generating foundational maps for several tissues obtained from normal individuals across a wide range of ages. A previous NIH-sponsored project, GTEx 5 , examined DNA variants and bulk tissue expression patterns across approximately a thousand individuals, but HuBMAP is a distinct project focused on generating molecular maps that are spatially resolved at the single-cell level but using samples from a more limited number of people. To achieve these goals, HuBMAP has been designed as a cohesive and collaborative organization, with a culture of openness and sharing using team science-based approaches 6. The HuBMAP Consortium (https://hubmapconsortium.org/) will actively work with other ongoing initiatives including the Human Cell Atlas 7 , Human Protein Atlas 8 , LIfeTime (https://lifetime-fetflagship.eu/), and related NIH-funded consortia that are mapping specific organs (including the brain 9 , lungs (https://www.lungmap.net/), kidney (https...