Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.

Background: Activin receptor-like kinase 1 (ALK1) is an important regulator of normal blood vessel formation and pathological tumor angiogenesis. Results: Crystal structure of ALK1-BMP9-ACTRIIB signaling complex and kinetic and thermodynamic properties of receptorligand interactions are described. Conclusions: ALK1 achieves high specificity for BMP9/10 through unusual receptor positioning and unique receptor-ligand interface. Significance: Structural and mechanistic insights into ALK1 signaling provide a framework for novel anti-angiogenic therapies.

The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded ␤-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hingelike motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins (1). The domain is a small three-stranded ␤-sheet stabilized by the stacking of several conserved aromatic and proline residues (2). Differences in residue identity at the binding surface result in a variation in ligand specificity that is used as the basis to divide WW domains into groups. For example, in group I WW domains that bind PPXY sequences (3), the Tyr is a key specificity residue and is accommodated by a largely hydrophobic pocket on the concave binding surface consisting of conserved Ile (or Val/Leu), His, and Gln (or Arg/Lys) residues. The Pro residues of the ligand contribute to the binding by stacking against the Trp and Tyr residues that form a second interaction site (4). It is evident that, since a number of proteins are likely to contain WW domain recognition sites, further factors most probably contribute to increasing affinity and specificity of a WW domain for a target. For example, the Pro-rich sequence in ␤-dystroglycan targeted by the dystrophin WW domain requires a composite binding surface provided by the WW domain and an adjacent EF hand (4). The binding of murine Nedd4 to the amiloride-sensitive epithelial sodium channel requires direct involvement of two of its three WW domains (5). Indeed, WW domains often exist in multiple numbers within a protein. Multiple modules may act in concert to achieve greater specificity for a target ...

IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other β-trefoil family proteins in the IL-1 and FGF families.T he IL-1 cytokines (IL-1α and IL-1β) are master mediators of inflammatory responses (1). IL-1β also regulates immune function through its role in T helper 17 (Th17) cell differentiation and maintenance (2, 3). IL-1 action has been implicated in numerous human diseases, including rheumatoid arthritis, MuckleWells syndrome, gout, type 2 diabetes, and stroke (4). Several natural mechanisms directly oppose the actions of IL-1, including a soluble and cell surface decoy receptor (IL-1R2), a natural antagonist (IL-1Ra), and a soluble signaling receptor (IL-1R1) (5). Therapeutics that block IL-1 based on these mechanisms have been developed (6-8).Recently, a nonoptimized formulation of anakinra (methionyl-IL-1Ra; Kineret) was shown to provide clinical benefit in dry-eye disease (DED) (9). Moderate to severe DED is a chronic inflammatory condition of the corneal surface that results in pain, discomfort, and epitheliopathy (as measured by fluorescein staining). Inability to maintain a proper tear film over the cornea (owing to a variety of etiologies) results in desiccating stress, which drives an inflammatory cascade (10, 11). IL-1 plays a central role in the initiation and maintenance of this cascade, as well as in the pain mediated by the corneal neural plexus. IL-1α and IL-1β protein are elevated in the lacrimal gland, tears, and the ocular surface in all forms of dry-eye disease (12), and their mRNA is increased in both humans and in rodent disease models (13,14). Genetic ablation of IL-1R1, the primary receptor for IL-1α and IL-1β, can block the development of corneal staining in a Sjögren syndrome corneal epitheliopathy model (15), and topically administered anakinra can improve surface epithliopathy in a mouse dry-eye model (14). IL-1β is essential for Th17 cell differentiation and maintenance, and Th17 cells are likely the main effector cells that induce epithelial damage (2, 3). Genetic and pharmacologic studies have shown that IL-1β mediates, and IL-1Ra blocks, normal, inflamm...

DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of “undruggability” for an intracellular target. Structural studies reveal extensive protein–WDB002 and protein–protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise “undruggable” targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.

The type II restriction endonuclease BstYI recognizes the degenerate sequence 5'-RGATCY-3' (where R = A/G and Y = C/T), which overlaps with both BamHI (GGATCC) and BglII (AGATCT), and thus raises the question of whether BstYI DNA recognition will be more BamHI-like or BglII-like. We present here the structure of BstYI bound to a cognate DNA sequence (AGATCT). We find the complex to be more BglII-like with similarities mapping to DNA conformation, domain organization, and residues involved in catalysis. However, BstYI is unique in containing an extended arm subdomain, and the mechanism of DNA capture has both BglII-like and BamHI-like elements. Further, DNA recognition is more minimal than BglII and BamHI, where only two residues mediate recognition of the entire core sequence. Taken together, the structure reveals a mechanism of degenerate DNA recognition and offers insights into the possibilities and limitations in altering specificities of closely related restriction enzymes.