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1.  Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution 
The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101.
Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2NTKD, has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-­O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2NTKD with a dissociation constant (K d) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K d for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca2+.
PMCID: PMC3565440  PMID: 23385462
protein kinases; inhibitors; flavonol glycosides; quercitrin; SL0101
2.  Application of protein engineering to enhance crystallizability and improve crystal properties 
This article presents an overview of protein-engineering methods designed to enhance crystallizability and discusses a number of examples of their successful application.
Until recently, protein crystallization has mostly been regarded as a stochastic event over which the investigator has little or no control. With the dramatic technological advances in synchrotron-radiation sources and detectors and the equally impressive progress in crystallographic software, including automated model building and validation, crystallization has increasingly become the rate-limiting step in X-ray diffraction studies of macromolecules. However, with the advent of recombinant methods it has also become possible to engineer target proteins and their complexes for higher propensity to form crystals with desirable X-ray diffraction qualities. As most proteins that are under investigation today are obtained by heterologous overexpression, these tech­niques hold the promise of becoming routine tools with the potential to transform classical crystallization screening into a more rational high-success-rate approach. This article presents an overview of protein-engineering methods designed to enhance crystallizability and discusses a number of examples of their successful application.
PMCID: PMC3089013  PMID: 20445236
protein engineering
3.  It’s all in the crystals… 
Protein surface engineering is increasingly used as a routine tool to enhance the crystallization propensity of proteins. Future possibilities include the use of multi-site protein variants, rational modulation of solubility and the development of strategies to tackle membrane proteins.
Macromolecular crystallography relies on the availability and quality of single crystals; these are typically obtained through extensive screening, which has a very low intrinsic success rate. Crystallization is not a completely stochastic process and many proteins do not succumb to crystallization because of specific microscopic features of their molecular surfaces. It follows that rational surface engineering through site-directed mutagenesis should allow a systematic and significant improvement in crystallization success rates. Here, one such established strategy, surface-entropy reduction (SER), is discussed, including its successes, limitations and possible future developments.
PMCID: PMC3069739  PMID: 21460442
macromolecular crystallography; crystals; crystallization; protein engineering; crystal contacts
4.  The role of entropy and polarity in intermolecular contacts in protein crystals 
Logistic regression was used to study the amino-acid composition and structure of crystal contacts in monomeric proteins. Crystal contacts are generally depleted of large flexible amino acids and enriched in small and hydrophobic residues such as Gly and Leu; additionally, larger contacts have cores depleted of polar residues.
The integrity and X-ray diffraction quality of protein crystals depend on the three-dimensional order of relatively weak but reproducible intermolecular contacts. Despite their importance, relatively little attention has been paid to the chemical and physical nature of these contacts, which are often regarded as stochastic and thus not different from randomly selected protein surface patches. Here, logistic regression was used to analyze crystal contacts in a database of 821 unambiguously monomeric proteins with structures determined to 2.5 Å resolution or better. It is shown that the propensity of a surface residue for incorporation into a crystal contact is not a linear function of its solvent-accessible surface area and that amino acids with low exposed surfaces, which are typically small and hydrophobic, have been underestimated with respect to their contact-forming potential by earlier area-based calculations. For any given solvent-exposed surface, small and hydrophobic residues are more likely to be involved in crystal contacts than large and charged amino acids. Side-chain entropy is the single physicochemical property that is most negatively correlated with the involvement of amino acids in crystal contacts. It is also shown that crystal contacts with larger buried surfaces containing eight or more amino acids have cores that are depleted of polar amino acids.
PMCID: PMC2672819  PMID: 19390155
intermolecular contacts; entropy; polarity
5.  Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains 
Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described.
The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR_C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni2+ ions but that it is able to bind Zn2+ with K d < 70 nM. It is concluded that Zn2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.
PMCID: PMC2659884  PMID: 19307717
transcription regulation; GntR family; structural genomics; surface-entropy reduction
6.  Harvesting the high-hanging fruit: the structure of the YdeN gene product from Bacillus subtilis at 1.8 Å resolution 
High-throughput (HT) protein crystallography is severely impeded by the relatively low success rate of protein crystallization. Proteins whose structures are not solved in the HT pipeline owing to attrition in any phase of the project are referred to as the high-hanging fruit, in contrast to those proteins that yielded good-quality crystals and crystal structures, which are referred to as low-hanging fruit. It has previously been shown that proteins that do not crystallize in the wild-type form can have their surfaces engineered by site-directed mutagenesis in order to create patches of low conformational entropy that are conducive to forming intermolecular interactions. The application of this method to selected proteins from the Bacillus subtilis genome which failed to crystallize in the HT mode is now reported. In this paper, the crystal structure of the product of the YdeN gene is reported. Of three prepared double mutants, i.e. E124A/K127A, E167A/E169A and K88A/Q89A, the latter gave high-quality crystals and the crystal structure was solved by SAD at 1.8 Å resolution. The protein is a canonical α/β hydrolase, with an active site that is accessible to solvent.
PMCID: PMC2792027  PMID: 15159570

Results 1-6 (6)