Evaluation of integrin αvß6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.

High-density grids for efficient data collection from multiple crystals.

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

Cleaning protocols for crystallization robots: preventing protease contamination.

The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5 mM. Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1 M. Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1 M NaOH, as for example specified in the original ZENM protocol, are recommended to completely deactivate Zymit protease activity.

High-resolution crystal structures and spectroscopy of native and compound I cytochrome c peroxidase.

Cytochrome c peroxidase (CCP) is a 32.5 kDa mitochondrial intermembrane space heme peroxidase from Saccharomyces cerevisiae that reduces H(2)O(2) to 2H(2)O by oxidizing two molecules of cytochrome c (cyt c). Here we compare the 1.2 A native structure (CCP) with the 1.3 A structure of its stable oxidized reaction intermediate, Compound I (CCP1). In addition, crystals were analyzed by UV-vis absorption and electron paramagnetic resonance spectroscopies before and after data collection to determine the state of the Fe(IV) center and the cationic Trp191 radical formed in Compound I. The results show that X-ray exposure does not lead to reduction of Fe(IV) and only partial reduction of the Trp radical. A comparison of the two structures reveals subtle but important conformational changes that aid in the stabilization of the Trp191 cationic radical in Compound I. The higher-resolution data also enable a more accurate determination of changes in heme parameters. Most importantly, when one goes from resting state Fe(III) to Compound I, the His-Fe bond distance increases, the iron moves into the porphyrin plane leading to shorter pyrrole N-Fe bonds, and the Fe(IV)-O bond distance is 1.87 A, suggesting a single Fe(IV)-O bond and not the generally accepted double bond.

Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential.

Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.

Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP).

We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer. In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+). The crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing [K(+)] and the lifetime of the Trp191 radical also has been considerably shortened in this mutant. This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing [K(+)] when horse heart cytochrome c is the substrate. However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength. We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.

Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.