Publications

We show that the three conformational states of integrin αβ have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact αβ On the surface of K562 cells, αβ is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a β-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.

Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.

Bacteria and fungi in shared environments compete with one another for common substrates, and this competition typically involves microbially-produced small molecules. An investigation of one shared environmental niche, the carton material of the Formosan subterranean termite Coptotermes formosanus, identified the participants on one of these molecular exchanges. Molecular characterization of several termite-associated actinobacteria strains identified eleven known antimicrobial metabolites that may aid in protecting the C. formosanus colony from pathogenic fungal infections. One particular actinobacterial-derived small molecule, bafilomycin C1, elicited a strong chemical response from Trichoderma harzianum, a common soil saprophyte. Upon purification and structure elucidation, three major fungal metabolites were identified, t22-azaphilone, cryptenol, and homodimericin A. Both t22-azaphilone and homodimericin A are strongly upregulated, 123- and 38-fold, respectively, when exposed to bafilomycin C1, suggesting each play a role in defending T. harzianum from the toxic effect of bafilomycin C1.

The microtubule cytoskeleton is the structural base of key cellular processes such as axonal growth, intracellular transport and cell division. These carefully orchestrated processes rely on the precise control of microtubule function and architecture. Microtubules, however, are dynamic polymers that stochastically transition between periods of growth and shrinkage. Therefore, the cell must regulate microtubule dynamics to achieve control over the length and stability of the microtubule cytoskeleton to accurately perform its cellular functions. Kinesins are best known for their ability to move processively on microtubules and carry cargo. Nevertheless, some kinesin families can also regulate microtubule dynamics and organize microtubule structures. Kinesin-8s play a central role in regulating the microtubule cytoskeleton by controlling microtubule length. Although extensive studies have been conducted, the mechanism by which kinesin-8 disassemble microtubules has remained unclear. My thesis work identified the central features of this mechanism for the yeast kinesin-8/Kip3. Like other kinesins, kinesin-8/Kip3, uses ATP hydrolysis for stepping on the microtubule lattice. Upon binding to curved tubulin at the plus-end, Kip3 undergoes a switch in activity resulting in strong tubulin binding, thereby promoting microtubule depolymerization. Our model is supported by the identification of the structural elements in the motor domain and in tubulin required for plus-end recognition, long-range processivity and microtubule depolymerization by Kip3. To obtain a comprehensive understanding of the Kip3-tubulin interaction we obtained high-resolution structures using cryo-electron microscopy of Kip3 bound to stabilized microtubules. This approach identified intra and intermolecular interactions that underlie Kip3’s ability to switch from a motile kinesin to a depolymerase at the microtubule end. The coexistence of the two activities in kinesin-8/Kip3, gives rise to its emergent property of length-dependent microtubule disassembly. Another challenge in the regulation of microtubule structures is in the control of the nucleation and architecture of the microtubule polymer. To overcome these challenges we developed a programmable DNA-origami microtubule seed, which can template defined microtubule structures. This tool will enable future studies on the regulation of microtubule architecture, such as the microtubule doublet structures found in cilia. Together, these findings elucidate regulatory mechanisms whereby nanometer-size molecules can measure and regulate micron-sized cellular microtubule structures.

The bacterial chromosome is dramatically compacted into a subcellular structure called nucleoid. Simultaneously, it must be organized to allow the protein machinery for essential cellular processes to gain access to the DNA. Bacteria lack the histones that play a critical role in chromosome compaction in other kingdoms of life; instead they have evolved other mechanisms to compact, organize, and segregate their chromosomes. Particularly, a group of chromosomal architectural proteins called nucleoid-associated proteins (NAPs) and the highly conserved structural maintenance of chromosomes (SMC) condensin complex are two of the major players in bacterial chromosome condensation and organization. Emerging single-molecule techniques, which can detect transient changes in DNA structure with high spatial and temporal resolutions, have contributed significantly to our understanding of how NAPs and SMC family members condense DNA. However, most nanomanipulation methods only measure the change in extension of DNA as a proxy to investigate protein-DNA interactions; they are unable to directly measure protein binding on DNA without fluorescently labeling the protein. In this dissertation, I describe a new approach to simultaneously monitor the association of proteins with DNA and changes in DNA conformation, while bypassing the difficult procedure of labeling proteins. Our method is demonstrated to be a relatively simple yet highly quantitative assay to study any DNA-binding protein in general. The second part of this dissertation focuses on the study of ParB, which is part of the parABS partitioning system required in chromosome segregation for most bacteria. ParB binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA in a phenomenon called spreading. Previous studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystalized with parS revealed that ParB dimers form a higher order tetrameric complex. Using this structure as a guide, a series of proposed intermolecular interactions in the Bacillus subtilis ParB was ablated to investigate their effect on spreading. Our results, based on in vivo and in vitro characterizations, demonstrated that a network of both cis and trans interactions between ParB dimers is necessary for spreading.

Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

Trypanosomes that cause sleeping sickness endocytose apolipoprotein L1 (APOL1)-containing trypanolytic factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome Trypanosoma brucei rhodesiense counteracts trypanolysis by expressing the surface protein serum response-associated (SRA), which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C terminal predicted coiled-coil (CC) domains of human APOL1 G1 (S342G/I384M) and G2 (ΔN388Y389) decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, thus preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at a high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and molecular dynamics (MD) simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with the common variant G0. Conformational plasticity in G1 and G2 CC domains led to interhelical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered the structure of both helix α2 and helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease.

Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In , heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore 'interrupted' with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading.

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin's shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin's biosynthesis including a subcluster of genes consistent with selvamicin's 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

Extracts of Alzheimer's disease (AD) brain that contain what appear to be sodium dodecyl sulfate-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated the Aβ monomer, consist primarily of Aβ42, and resist denaturation by chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in two ways: by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then used a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by the Aβ monomer. The results suggest that the cross-links disfavor fibril formation from Aβ dimers, thereby enhancing the concentration of soluble aggregates akin to those in aqueous extracts of AD brain. Thus, it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates.

Mitochondrial sirtuins, SIRT3-5, are NAD-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.

Kinetochores connect centromeric nucleosomes with mitotic-spindle microtubules through conserved, cross-interacting protein subassemblies. In budding yeast, the heterotetrameric MIND complex (Mtw1, Nnf1, Nsl1, Dsn1), ortholog of the metazoan Mis12 complex, joins the centromere-proximal components, Mif2 and COMA, with the principal microtubule-binding component, the Ndc80 complex (Ndc80C). We report the crystal structure of Kluyveromyces lactis MIND and examine its partner interactions, to understand the connection from a centromeric nucleosome to a much larger microtubule. MIND resembles an elongated, asymmetric Y; two globular heads project from a coiled-coil shaft. An N-terminal extension of Dsn1 from one head regulates interactions of the other head, blocking binding of Mif2 and COMA. Dsn1 phosphorylation by Ipl1/Aurora B relieves this autoinhibition, enabling MIND to join an assembling kinetochore. A C-terminal extension of Dsn1 recruits Ndc80C to the opposite end of the shaft. The structure and properties of MIND show how it integrates phospho-regulatory inputs for kinetochore assembly and disassembly.

The immune checkpoint receptor PD-1 and its ligand, PD-L1, have emerged as key regulators of anti-tumor immunity in humans. Recently, we reported an ultra-high-affinity PD-1 mutant, termed high-affinity consensus (HAC) PD-1, which shows superior therapeutic efficacy in mice compared with antibodies. However, the molecular details underlying the action of this agent remain incompletely understood, and a molecular view of PD-1/PD-L1 interactions in general is only beginning to emerge. Here, we report the structure of HAC PD-1 in complex with PD-L1, showing that it binds PD-L1 using a unique set of polar interactions. Biophysical studies and long-timescale molecular dynamics experiments reveal the mechanisms by which ten point mutations confer a 35,000-fold enhancement in binding affinity, and offer atomic-scale views of the role of conformational dynamics in PD-1/PD-L1 interactions. Finally, we show that the HAC PD-1 exhibits pH-dependent affinity, with pseudo-irreversible binding in a low pH setting akin to the tumor microenvironment.

Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A's formation by this path, a six-electron oxidation, could be a response to oxidative stress triggered by bafilomycin C1.

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.

Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families.

The Wnt/β-catenin signaling pathway plays a major role in tissue homeostasis, and its dysregulation can lead to various human diseases. Aberrant activation of β-catenin is oncogenic and is a critical driver in the development and progression of human cancers. Despite the significant potential of targeting the oncogenic β-catenin pathway for cancer therapy, the development of specific inhibitors remains insufficient. Using a T cell factor (TCF)-dependent luciferase-reporter system, we screened for small-molecule compounds that act against Wnt/β-catenin signaling and identified MSAB (methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate) as a selective inhibitor of Wnt/β-catenin signaling. MSAB shows potent anti-tumor effects selectively on Wnt-dependent cancer cells in vitro and in mouse cancer models. MSAB binds to β-catenin, promoting its degradation, and specifically downregulates Wnt/β-catenin target genes. Our findings might represent an effective therapeutic strategy for cancers addicted to the Wnt/β-catenin signaling pathway.

Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.