Macromolecular Modeling Blog ™

In a recent Nature paper Koder & Anderson et al. describe the procedure for creating an O2 transport protein from first principals. The paper is somewhat technical and may appeal to the biochemists amongst our readers, here we present the main ideas, findings and conclusions. 

Throughout the paper, one of the main arguments of the authors is that due to the inherent complexity built into naturally occurring proteins, incurred by natural selection and evolution, it is harder to design a function or manipulate a complex natural protein, and would be easier to engineer a new one from scratch. They propose the “maquette approach”: develop–simple models that are progressively altered to test and determine the ultimate characteristics of the construction.

The outline for the design process is the following:

1. assembly of a simple, robust, generic protein framework, such as a helical bundle, of appropriate size to sustain eventual cofactor binding and catalytic function; 
2. insertion of cofactor-binding amino acids, keeping the number of amino acid changes low to control complexity; 
3. adjusting the sequence for improved structural resolution; 
4. iteratively testing, redesigning and adding engineering elements to refine function. Assembly of scaffold and cofactors; 

The actual design stages to construct the O2 transport protein included:

• Starting maquette comprising polar glutamate and lysine, as well as non-polar leucine. These have high a-helix-forming propensities which, when arranged in a near-repeating heptad sequence LEELLKK LEELLKL, spontaneously assemble into a water-soluble four-a-helical bundle with glutamate and lysine exposed and leucine buried in a molten globular interior. 
• Adding an amino-terminal CGGG sequence for disulphide-mediated dimerization restricts helical topologies to syn or anti. Completing this stage, an internal leucine at e-position 7 of each helix was replaced with tryptophan to facilitate optical detection of the protein. 
• In the second stage of design, replacing leucines at internal a-positions 10 and 24 with histidine was sufficient to anchor up to four haems in the bundle. The authors also replaced a-position leucine 17 between the haems with phenylalanine, an amino acid commonly found near haems in natural proteins. To allow discrimination between the haem-binding sites, they replaced another interior leucine with arginine.
• Singular structures in these bundles can be engineered without computation; introduction of b-branched aliphatic or aromatic residues along with a polar bond across helices provided by the histidines confers tertiary structure to the four-a-helix interior of the apo protein as seen by NMR and X-ray crystallography. 
• For oxygen transport the design was simplified by lowering the haem capacity from four to two by replacing histidine at position 24 with phenylalanine. Inspection of model apo structures showed that a substantial rotation of 50 deg. around the helical axes was required on haem binding to accommodate the histidine rotamers typical of natural bis-histidine haem-binding proteins. This rotation exports hydrophobic interior residues into, and imports polar residues from, the aqueous phase. 
• Modeling identified four amino acids for substitution to interfacially compatible alanines or glutamines and one for deletion to more favourably realign the binary pattern after haem addition. Three inwardly rotating b-position glutamates at positions 11, 18 and 25 were deliberately left in place to apply strain to weaken one of the two histidine-haem iron ligation interactions, as occurs in neuroglobin. 
• To ease NMR structure determination and assignment of 90% of the peptide backbone, external residues were also diversified at this time.
• Crystal structures and NMR of the intermediate (“still in design”) protein showed a high degree of inter-helix motion. Indeed, To constrain motion, the loops were reconfigured to link the helices across the most mobile interface. This also allowed the loops to be linked into a monomeric ‘candelabra’ structure. NMR confirmed the water-restricting effects of loop reconfiguration and monomerization. 

Results:

The protein binds heam:

The final design display ferric and ferrous haem visible spectra indicative of six-coordinate bis-histidine ligated haem B, characteristic of cytochrome b, deoxy-neuroglobin and cytoglobin, and quite distinct from the five-coordinate myoglobin and haemoglobin. 

Dissociation constant (Kd) for binding of the first haem (1 nM) is much tighter than that for the second haem (50 nM). NMR assignments unambiguously identified the first haem B to bind at H7 positions at the open end of the candelabra structure. Only ferrous haem shows rapid and complete conversion of the ferrous haem into the oxy-ferrous haem with a half time of ,50 ms measured by stopped-flow spectroscopy. This oxy-ferrous spectrum is remarkably similar to that of native neuroglobin.

The protein binds O2 better than CO:

• Kd of O2: 32 nM
• Kd of CO: 36 nM.

This is important since in all natural haemoglobins with distal histidines, either preferentially bound to the haem iron, as in neuroglobin, or displaced from the iron, as in myoglobin or human haemoglobin, CO is a poison that binds more tightly than O2.

Whereas the oxygen off-rate is similar to that of human neuroglobin, the on-rate is almost 100 times slower. and resembles that of Ascaris haemoglobin. The hypothesis is that a large hydrophobic pocket, as in neuroglobin, can speed O2 binding, whereas proximal strain, as in Ascaris haemoglobin, slows O2 binding. 

Helical strain confered by burial of glutamates promotes CO and O2 binding:

To test whether the helical rotation model of histidine strain was indeed operating to promote CO and O2 binding, the b-position glutamates was changed to alanines. As anticipated, CO binding slowed by more than an order of magnitude. Moreover, O2 failed to form detectable oxy-ferrous haem. 

One of these key engineering principles is the creation and control of protein motion. The long-range helical motion and strain that facilitates ligand exchange for oxygen transport, while still permitting relatively secure haem binding by means of bis-histidine ligation, would not have been possible merely by targeting the static structure of the final oxy-ferrous state in a natural haem protein.

Haem and substrate specificity: 

The interior of the design protein adopts a unique structure not only around haem B but also around other porphyrins including haem A. Haem A has markedly different peripheral substitutions, which result in different redox and spectral properties. 

Our take:

This beautiful work comes in succession to several previous works that were able to design either a novel protein fold from scratch, or a novel enzyme. The major difference between the approaches is that while this work preaches for simplicity and abstraction, e.g.: 

The fact that such a modest redesign succeeds supports the view that many protein-engineering elements do not require atomistic precision and that exacting mimics of natural protein sites is neither necessary nor good engineering.

The previous works relied heavily on an atomic resolution energy function and atomic level modeling. The question asked is: what would be the general solution? i.e. the globin O2 transport system is a very well known and studied system. Will the same simple design principles work for a system we know much less about? Many of the ‘first principles’ in the design are borrowed from nature. How would we be able to reproduce the approach in a system in which we don’t know what were nature’s solutions? 

In any way this is another important milestone on our road to understand protein structure-function relationship.

Koder, R., Anderson, J., Solomon, L., Reddy, K., Moser, C., & Dutton, P. (2009). Design and engineering of an O2 transport protein Nature, 458 (7236), 305-309 DOI: 10.1038/nature07841