Susanne Gräslund

In situ proteolysis for protein crystallization and structure determination

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.

A roadmap to generate renewable protein binders to the human proteome

Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.

The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins.

Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials.

Crystal Structure of Human Cytosolic 5′-Nucleotidase II INSIGHTS INTO ALLOSTERIC REGULATION AND SUBSTRATE RECOGNITION

Cytosolic 5′-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adenine-based nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5′-nucleotidase II as native protein (2.2 Å) and in complex with adenosine (1.5Å) and beryllium trifluoride (2.15Å). The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5′(3′)-deoxyribonucleotidase and cytosolic 5′-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5′-nucleotidase II with that of mitochondrial 5′(3′)-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.

Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display

To demonstrate the utility of phage display in generating highly specific antibodies, affinity selections were conducted on 20 related Src Homology 2 (SH2) domains (ABL1, ABL2, BTK, BCAR3, CRK, FYN, GRB2, GRAP2, LYN, LCK, NCK1, PTPN11 C, PIK3R1 C, PLCγ1 C, RASA1 C, SHC1, SH2D1A, SYK N, VAV1 and the tandem domains of ZAP70). The domains were expressed in Escherichia coli, purified and used in affinity selection experiments. In total, 1292/3800 of the resultant antibodies were shown to bind the target antigen. Of the 695 further evaluated in specificity ELISAs against all 20 SH2 domains, 379 antibodies were identified with unique specificity (i.e. monospecific). Sequence analysis revealed that there were at least 150 different clones with 1–19 different antibodies/antigen. This includes antibodies that distinguish between ABL1 and ABL2, despite their 89% sequence identity. Specificity was confirmed for many on protein arrays fabricated with 432 different proteins. Thus, even though the SH2 domains share a common three-dimensional structure and 20–89% identity at the primary structure level, we were able to isolate antibodies with exquisite specificity within this family of structurally related domains.

Crystal Structure of Human Inosine Triphosphatase: Substrate Binding and Implication of the Inosine Triphosphatase Deficiency Mutation P32T.

Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1Å, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer.

Apollo-NADP+: a spectrally tunable family of genetically encoded sensors for NADP+

NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP+ redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP+ (Apollo-NADP+) that exploits NADP+-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose-stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP+ to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean-tagged version of Apollo-NADP+ with the H2O2 sensor HyPer.

A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

A pilot project to generate affinity reagents to human proteins

acKnoWLedGments We thank all members of the Structural Biology Center and Midwest Center for Structural Genomics at Argonne National Laboratory for their help in conducting these experiments, S. Li, C.B. Lindberg and C. Giometti for running mass spectrometry experiments, A. Edwards for reading and commenting on the drafts, and L. Butler for preparing the manuscript. This work was supported by US National Institutes of Health (GM62414, GM074942) and by the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.