The long road to an elusive structure

From the Stoddard lab, Basic Sciences Division

Restriction endonucleases (REases) function as an innate form of microbial immune systems to disrupt invasive DNA molecules, such as viral genomes, that infect bacterial and archaeal cells. Target sequences cleaved by REases are recognized through protein-DNA interactions, and are intrinsic to the structure of each enzyme. Restriction endonucleases are ubiquitous across the prokaryotic universe and are constantly in flux as part of an ongoing ‘arms race’ between their hosts and potential invaders (typically bacteria and phage). Their activity is extraordinarily fine-tuned to target exact DNA recognition sites: most REases display complete loss of cleavage activity in response to alteration of a single base pair in their targets.

The most commonly employed and best understood REases are usually obligate dimers and use two copies of the same catalytic site to cleave the each of the strands of the DNA target; Type IIT REases are an exception, and use different catalytic sites to cleave each strand of the DNA target site.

Basic Sciences member Dr. Barry Stoddard and his laboratory have long been fascinated by REases and bacterial restriction-modification systems, both in terms of their evolutionary history and their mechanisms of DNA recognition and fidelity.  Dr. Stoddard’s lab is the first to report the structure of a representative Type IIT enzyme, and published their findings in the journal Nucleic Acids Research.

The authors picked the best characterized of the Type IIT enzyme, BbvCI. Dr. Stoddard explained why the BbvCI enzyme represents a fascinating ‘missing link’ (or ‘crazy cousin’) within the restriction endonuclease family tree.  “Most REases of its type are either homodimers (constructed of two identical enzyme subunits, that recognize and cleave a palindromic DNA target site) or gene-fused, pseudo-symmetric monomers (constructed of a single protein chain containing two consecutive, closely related enzyme domains, that recognize and cleave a non-palindromic DNA target).  BbvCI is an outlier: it’s constructed out of two non-identical enzyme subunits, that come together to form an enzyme assemblage that acts on a completely asymmetric target.”  


Structure of BbvCI tetrameric assemblage Figure from Dr. Stoddard

To determine the structural organization of BbvCI, and the basis for asymmetric recognition and enzymatic activity, the authors identified its remaining catalytic site residues and crystalized the intact enzyme.

It turned out to be the longest and most challenging study undertaken in the Stoddard lab in terms of years invested in solving the structure. “We first started working on this enzyme in the year 2000, and finally published the structure 19 years later…that’s almost 3/4ths of the entire history of my lab’s presence at the Center!” said Dr. Stoddard. To solve the structure, Dr. Betty Shen, a senior staff scientist in the Stoddard lab had to resort to some classic old-fashioned ‘crystallographer’s tricks’, combined with modern approaches for phasing her X-ray data. She collected over 100 individual X-ray diffraction data sets on various crystals of the enzyme.

Dr. Stoddard: “The project was further complicated by the fact that the composition and behavior of the enzyme was far more complex than we expected -- it was found to be in the form of a tetramer, with two copies of each subunit, rather than the expected heterodimer; one of the two subunits is easily dissociated from its sibling and lost during purification and crystallization.” 

The determination of the structure (and a careful analysis of the behavior of the enzyme during the entire process of solving that structure) eventually revealed many details of its form and function, and led the authors to more accurately place it within an evolutionary scheme that relates the various types of REase architectures. The solution behavior of the enzyme was examined to account for its atypical behavior during crystallization. This was further investigated using tethered, single-chain enzyme variants to prevent dissociation. To investigate the DNA-recognition ability of the enzyme, the authors performed homology-based modeling and computational docking utilizing mutation of specific residues.

As for what lies ahead after this breakthrough, Dr. Stoddard has many plans up his sleeve.  “We are moving on to structural analyses of far larger and more complex restriction-modification systems, including one that brings together multiple copies of several protein subunits harboring DNA recognition, DNA methylation, and DNA cleavage activities. The mass of that intact enzyme assemblage is over a half-million daltons. Determination of its structure (in the presence and absence of bound DNA) will require the use of Cryo-electron microscopy (‘Cryo-EM’), which Betty is now starting to learn and use. The project is also serving as our ‘learning system’ as we work to eventually bring CryoEM expertise and instrumentation to the Center, as part of positioning the entire structural biology program for the next 20 years of research.” said Dr. Stoddard.

Shen BW, Doyle L, Bradley P, Heiter DF, Lunnen KD, Wilson GG, Stoddard BL. 2018. Structure, subunit organization and behavior of the asymmetric Type IIT restriction endonuclease BbvCI. Nucleic Acids Res, Vol 47, No. 1

This work was supported by the National Institute for General Medical Sciences, Fred Hutchinson Cancer Research Center and New England Biolabs.