The inhibition of mutant KRAS proteins has emerged as a promising approach for treating KRAS-driven cancers, as evidenced by the clinical success of KRAS G12C inhibitors. KRAS G12D, the most common mutant, promises significant expansion of the addressable patient population; however, the reduced nucleophilicity of aspartate compared to cysteine poses significant challenges in balancing sufficient potency with ADME properties to support oral exposure. Herein, we describe the discovery of KRAS G12D inhibitor 23 (INCB159020), which achieves oral exposure in nonhuman primate (NHP). Starting from a weakly potent hit, structure-based drug design was utilized to drive significant potency. Focus on molecular rigidity and balanced polarity then allowed for successful optimization of properties required for oral exposure.

CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.

Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.

Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations-an "extended" architecture and an "arch-shaped" architecture-although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragment (Fab) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Thus, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.

Models of target proteins in complex with small molecule ligands or peptide ligands are of significant interest to the biomedical research community. Structure-guided lead discovery and structure-based drug design make extensive use of such models. The bound ligands comprise only a small fraction of the total X-ray scattering mass, and therefore particular care must be taken to properly validate the atomic model of the ligand as experimental data can often be scarce. The ligand model must be validated against both the primary experimental data and the local environment, specifically: (1) the primary evidence in the form of the electron density, (2) examined for reasonable stereochemistry, and (3) the chemical plausibility of the binding interactions must be inspected. Tools that assist the researcher in the validation process are presented.

Polysaccharide monooxygenases (PMOs) are secreted metalloenzymes that catalyze the oxidative degradation of polysaccharides in a copper-, oxygen-, and reductant-dependent manner. Cellulose-active fungal PMOs degrade cellulosic substrates to be utilized as a carbon source for fungal growth. To gain insight into the PMO mechanism, the role of conserved residues in the copper coordination sphere was investigated. Here, we report active-site hydrogen-bonding motifs in the secondary copper coordination sphere of MtPMO3*, a C1-oxidizing PMO from the ascomycete fungus Myceliophthora thermophila. A series of point substitutions that disrupt this conserved network are used to interrogate its function. Activity assays, in conjunction with EPR spectroscopy, demonstrate that residues H161 and Q167 are involved in stabilizing bound oxygen, and H161 appears to play a role in proton transfer. Additionally, Q167 increases the ligand donor strength of Y169 to the copper via a hydrogen-bonding interaction. Altogether, H161 and Q167 are important for oxygen activation, and the results are suggestive of a copper-oxyl active intermediate.

Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.

Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.

Significance Maintenance and reprogramming of pluripotency are among the most important issues in stem cell biology and regenerative medicine. Pluripotency is governed by several key transcription factors regulating transcription of other factors. Among these, regulation of OCT4 transcription by NANOG (from Irish myth-ology Tír na nÓg) is a critical interaction. We present here the crystal structure of human NANOG homeodomain in complex with the OCT4 promoter DNA and, through a series of ration-ally designed mutations, we identify key functional residues in the protein–DNA interaction, protein stability, and maintenance of mouse ESC self-renewal. Furthermore, we describe a mutation, NANOG L122A, which enhances DNA binding affinity, protein stability, mouse ESC self-renewal, and reprogramming into ground state from primed state pluripotency.

The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.

The TM1088 locus of T. maritima codes for two proteins designated TM1088A and TM1088B, which combine to form the cytosolic portion of a putative Trk K+ transporter. We report the crystal structure of this assembly to a resolution of 3.45 Å. The high resolution crystal structures of the components of the assembly, TM1088A and TM1088B, were also determined independently to 1.50 Å and 1.55 Å, respectively. The TM1088 proteins are structurally homologous to each other and to other K+ transporter proteins, such as TrkA. These proteins form a cytosolic gating ring assembly that controls the flow of K+ ions across the membrane. TM1088 represents the first structure of a two-subunit Trk assembly. Despite the atypical genetics and chain organization of the TM1088 assembly, it shares significant structural homology and an overall quaternary organization with other single-subunit K+ gating ring assemblies. This structure provides the first structural insights into what may be an evolutionary ancestor of more modern single-subunit K+ gating ring assemblies.

X-ray crystallography provides the most accurate models of protein–ligand structures. These models serve as the foundation of many computational methods including structure prediction, molecular modelling, and structure-based drug design. The success of these computational methods ultimately depends on the quality of the underlying protein–ligand models. X-ray crystallography offers the unparalleled advantage of a clear mathematical formalism relating the experimental data to the protein–ligand model. In the case of X-ray crystallography, the primary experimental evidence is the electron density of the molecules forming the crystal. The first step in the generation of an accurate and precise crystallographic model is the interpretation of the electron density of the crystal, typically carried out by construction of an atomic model. The atomic model must then be validated for fit to the experimental electron density and also for agreement with prior expectations of stereochemistry. Stringent validation of protein–ligand models has become possible as a result of the mandatory deposition of primary diffraction data, and many computational tools are now available to aid in the validation process. Validation of protein–ligand complexes has revealed some instances of overenthusiastic interpretation of ligand density. Fundamental concepts and metrics of protein–ligand quality validation are discussed and we highlight software tools to assist in this process. It is essential that end users select high quality protein–ligand models for their computational and biological studies, and we provide an overview of how this can be achieved.

Abstract The crystallisation of proteins and macromolecular complexes is an essential step for studying the three‐dimensional structure of the molecular components that make up living cells. Protein crystals are formed by the self‐assembly of protein molecules into an ordered, periodic lattice arrangement. Crystallisation is initiated by reducing the solubility of the protein sample by the addition of precipitating agents such as salts and polyethylene glycols (PEG). This creates a supersaturated solution from which a protein‐rich phase will eventually separate. Under favourable conditions, the protein‐rich phase can adopt the form of an ordered protein crystal. The most common technique used for crystallising proteins is vapour diffusion, which relies on the loss of water from the growth solution during equilibration with a reservoir of precipitant. Protein crystals can be extremely difficult to grow and often require exhaustive screening of many different parameters such as concentration and type of precipitant, pH, temperature, additives and variations in protein construct. Key Concepts: A detailed knowledge of the molecular structure of proteins and macromolecular complexes is essential for understanding how the molecular machinery of living cells operates. Crystallography is the most prevalent method for studying the structures of proteins and macromolecular complexes at the heart of the cellular machinery. Protein crystals are formed by the self‐assembly of large polypeptide chains into an ordered, regular and repeating lattice arrangement of protein molecules. Crystallisation of proteins is generally carried out by the addition of precipitating agents to the protein solution, which results in the generation of a metastable, supersaturated solution from which a protein‐rich phase will eventually separate. Under favourable conditions, this protein‐rich phase can adopt the form an ordered protein crystal. Several experimental techniques are available to crystallise proteins, including vapour diffusion, microbatch crystallisation under oil, microdialysis, free‐interface diffusion and crystallisation in Lipidic Cubic Phase (LCP). The most commonly used crystallisation technique is vapour diffusion, which relies on the diffusion of water from the crystal growth solution to a precipitant reservoir. Even the most beautiful protein crystals do not necessarily diffract X‐rays. Many protein crystals suffer from disordered regions as a result of thermal or static displacement of atoms. Disorder is often a result of flexible domains in the protein that may adversely affect how the protein molecules are packaged together in the crystal. Recombinant DNA techniques can be used to engineer protein constructs that are more amenable to crystallisation by increasing the stability and/or solubility of the protein. Additionally, the amino acid residues on the surface of the protein can be modified to alter the intermolecular surface contacts within the crystal. Complexes of proteins bound to small molecule substrates such as cofactors or potential drugs can be crystallised. Additionally, very large complexes between multiple proteins or with other macromolecules such as RNA or DNA can be crystallised.

Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins and represents the first evidence that kanadaptin is likely involved in a phosphorylation-mediated signaling pathway. These results will be of use for designing experiments to further probe the function of kanadaptin.