Page, R. C. | bioRxiv Science

bioRxiv2018

A non-canonical metal center drives activity of the Sediminispirochaeta smaragdinae metallo-β- lactamase SPS-1

In an effort to evaluate whether a recently reported putative metallo-{beta}-lactamase (M{beta}L) contains a novel M{beta}L active site, SPS-1 from Sediminispirochaeta smaragdinae was over-expressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equivalents of Zn (II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not {beta}-lactam substrates with bulky substituents in the 6-7 position. Spectroscopic studies on Co (II)-substituted SPS-1 suggest a novel metal center in SPS-1, with reduced spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar that that in NDM-1 and other subclass B1 M{beta}Ls; however, the Zn1 metal ion is coordinated by 2 histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 [A] from the position reported in other M{beta}Ls. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents in the 6/7 position of {beta}-lactam antibiotics. This study reveals a novel metal binding site in M{beta}Ls, and suggests that the targeting of metal binding sites in M{beta}Ls with inhibitors is now more challenging with the identification of this new M{beta}L.

biochemistry↗

bioRxiv2018

Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of cerebellar CHIPopathy

CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. Mutations in CHIP are the driver of spinocerebellar autosomal recessive 16 (SCAR16), or cerebellar CHIPopathy, as we initially discovered this disease was caused by a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrate that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and alters the co-chaperone function of CHIP. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.

animal behavior and cognition↗