The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.chemical biology ͉ crystallography ͉ human S tructural, functional, and chemical genomics (proteomics) are disciplines that aim to determine the biochemical, cellular, and physiological functions of proteins on a genome scale. Many of the central, important experimental approaches that are involved, such as protein-based screens for small-molecule inhibitors, depend on the availability of purified and active proteins. To meet this demand, many large projects are devoted to developing methods to generate large numbers of purified proteins. However, the task is proving challenging: on average, for proteins from prokaryotes, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity (1, 2). The success rates for human proteins are predicted to be significantly lower.To improve the general rates of protein purification, efforts have focused largely on alterations of the recombinant host, the expression conditions, changes of the construct encoding the protein, and the purification conditions. It is also known that the expression and purification of a protein can be improved significantly by the addition of a specific ligand, which serves to stabilize the protein, thereby reducing its propensity to unfold, aggregate, or succumb to proteolysis. This parameter has not been studied systematically, although in individual cases the addition of a specific ligand has had dramatic effects. For example, the recombinant expression of the guinea pig and human forms of the enzyme 11␤-hydroxysteroid dehydrogenase-1 in bacteria was increased dramatically by the addition of an inhibitor of the enzyme to the growing cells (3) Wu, K. L. Kav...

Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

DEXD/H-box RNA helicases couple ATP hydrolysis to RNA remodeling by an unknown mechanism. We used x-ray crystallography and biochemical analysis of the human DEXD/H-box protein DDX19 to investigate its regulatory mechanism. The crystal structures of DDX19, in its RNA-bound prehydrolysis and free posthydrolysis state, reveal an ␣-helix that inserts between the conserved domains of the free protein to negatively regulate ATPase activity. This finding was corroborated by biochemical data that confirm an autoregulatory function of the N-terminal region of the protein. This is the first study describing crystal structures of a DEXD/H-box protein in its open and closed cleft conformations.RNA helicase activity is involved in all aspects of RNA metabolism, including transcription, pre-mRNA splicing, ribosome biogenesis, nuclear export, translation initiation and termination, RNA degradation, viral replication, and viral RNA detection. The DEXD/H-box RNA helicases couple hydrolysis of ATP to cycles of RNA binding and release that typically result in non-processive RNA duplex unwinding (1) or disruption of RNP 3 complexes (2, 3). These proteins interact in a non-sequence-specific manner with the phosphoribose backbone of single-stranded RNA. DEXD/H-box RNA helicases contain two ␣/␤-RecA-like domains that both feature conserved sequence motifs involved in RNA binding and ATP hydrolysis (4, 5). Accessory proteins are involved in the regulation of RNA binding and ATPase activities, although no general mechanism has been demonstrated.The DDX19 member of the DEXD/H-box RNA helicase family performs an essential function in mRNA nuclear export by remodeling RNP particles during passage of mRNA through the nuclear pore complex (3, 6). Dbp5, the yeast orthologue of DDX19 (7,8), causes displacement of the RNP constituent, Mex67, thereby preventing re-entry of mRNA into the nucleus (9). Dbp5 is also involved in translation termination (10). A specific function has been assigned to the ADP-bound form of Dbp5, which displaces the RNA-binding protein Nab2, an event that is required for mRNA export (3). In vivo, Dbp5 is activated by the nuclear pore complex-associated protein, Gle1 (11,12). Crystal structures of DEXD/H-box proteins show two-lobed proteins with the nucleotide binding site located in the lower part of the cleft separating the conserved domains and the RNA binding site across the upper cleft opening (13-17). DEXD/Hbox helicases in general share little homology in their coding sequences upstream of the conserved domain-1. The N-terminal extension of DDX19, however, shares significant homology with that of DDX25/GRTH, a testis-specific protein that is essential for spermatogenesis (18), supporting a functional significance for this sequence. Herein, we present a crystal structure of human DDX19 that shows the ADP-bound protein with an ␣-helical segment of the N-terminal extension wedged between the core domains, preventing cleft closure. In the structure of the ADPNP-bound protein in complex with RNA, this ␣-helix has moved...