Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant.

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.

Mechanism of the kinetically-controlled folding reaction of subtilisin.

Like many secreted proteases, subtilisin is kinetically stable in the mature form but unable to fold without assistance from its prodomain. The existence of high kinetic barriers to folding challenges many widely accepted ideas, namely, the thermodynamic determination of native structure and the sufficiency of thermodynamic stability to determine a pathway. The purpose of this article is to elucidate the physical nature of the kinetic barriers to subtilisin folding and to show how the prodomain overcomes these barriers. To address these questions, we have studied the bimolecular folding reaction of the subtilisin prodomain and a series of subtilisin mutants, which were designed to explore the steps in the folding reaction. Our analysis shows that inordinately slow folding of the mature form of subtilisin results from the accrued effects of two slow and sequential processes: (1) the formation of an unstable and topologically challenged intermediate and (2) the proline-limited isomerization of the intermediate to the native state. The low stability of nascent folding intermediates results in part from subtilisin's high dependence on metal binding for stability. Native subtilisin is thermodynamically unstable in the absence of bound metals. Because the two metal binding sites are formed late in folding, however, they contribute little to the stability of folding intermediates. The formation of productive folding intermediates is further hindered by the topological challenge of forming a left-handed crossover connection between beta-strands S2 and S3. This connection is critical to propagate the folding reaction. In the presence of the prodomain, folding proceeds through one major intermediate, which is stabilized by prodomain binding, independent of metal concentration and proline isomerization state. The prodomain also catalyzes the late proline isomerizations needed to form metal site B. Rate-limiting proline isomerization is common in protein folding, but its effect in slowing subtilisin folding is amplified because of the instability of the intermediate and an apparent need for simultaneous isomerization of multiple prolines in order to create metal site B. Thus, the kinetically controlled folding reaction of subtilisin, although unusual, is explained by the accrued effects of events found in other proteins.

Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin.

Residue-specific exchange rates of 223 amide protons in free and prodomain-complexed subtilisin were determined in order to understand how the prodomain binding affects the energetics of subtilisin folding. In free subtilisin, amide protons can be categorized according to exchange rate: 74 fast exchangers (rates > or = 1 h(-1)); 52 medium exchangers (rates between 1 h(-1) and 1 day(-1)); 31 slow exchangers (rates between 1 day(-1) and 0.001 day(-1)). The remaining 66 amide proteins did not exchange detectibly over 9 months (k(obs) < year(-1)) and were denoted as core protons. Core residues occur throughout the main structural elements of subtilisin. Prodomain binding results in high protection factors (100-1000) in the central beta-sheet, particularly in the vicinity of beta-strands S5, S6, and S7 and the connecting loops between them. These connecting loops provide the ligands to the cation at metal site B. Overall, prodomain binding seems to facilitate the organization of the entire central beta-sheet and alpha-helix C in the left-handed crossover connection between beta-strands two and three. It also appears to facilitate the isomerization of multiple prolines late in folding, allowing the formation of metal site B. The gain of stability region around site B comes at the cost of stability in regions more distal to prodomain binding: the C-terminal alpha-helix H and the N-terminal alpha-helices A and B. The acceleration of exchange in these regions by prodomain binding reveals an antagonism between the folding intermediate and the full native structure. This antagonism helps to explain why the prodomain is needed to stabilize the folding intermediate as well as why the unfolding of free subtilisin seldom occurs via this intermediate.

Main chain NMR assignments of subtilisin Sbt70 in its prodomain-bound state.

Main chain assignments are described for a 266-residue subtilisin mutant, Sbt70, in its 35 kDa complex with an N-terminal prodomain. The assignments provide the basis for understanding how the prodomain assists folding of subtilisin at a residue-specific level.

Directed coevolution of stability and catalytic activity in calcium-free subtilisin.

We have coevolved high activity and hyperstability in subtilisin by sequentially randomizing 12 amino acid positions in calcium-free subtilisin. The optimal amino acid for each randomized site was chosen based on stability and catalytic properties and became the parent clone for the next round of mutagenesis. Together, the 12 selected mutations increased the half-life of calcium-free subtilisin at elevated temperature by 15,000-fold. The catalytic properties of the mutants were examined against a range of substrates. In general, only mutations occurring at or near the substrate-binding surface have measurable effects on catalytic constants. No direct influence of stability on catalytic properties was observed. A high-stability mutant, Sbt140, was a more efficient enzyme in terms of k(cat)/K(m) than a commercial version of subtilisin across a range of substrates but had a lower k(cat) against tight-binding substrates. The reason for this behavior was discerned by examining microscopic rate constants for the hydrolysis of a tight-binding peptide substrate. Burst kinetics were observed for this substrate, indicating that acylation is not rate-limiting. Although acylation occurs at the rate of substrate binding, k(cat) is attenuated by the slow release of the N-terminal product. Natural evolution appears to have optimized catalytic activity against a range of sequences by achieving a balance between substrate binding and the rate of release of the N-terminal product.

Engineering subtilisin into a fluoride-triggered processing protease useful for one-step protein purification.

Subtilisin was engineered into a highly specific, processing protease, and the subtilisin prodomain was coengineered into an optimized recognition sequence. This involved five steps. First, a robust subtilisin mutant was created, which could tolerate the subsequent mutations needed for high specificity. Second, the substrate binding pocket was mutated to increase its sequence selectivity. Third, the subtilisin prodomain was engineered to direct cleavage to the junction of any protein fused to it. Fourth, the active site of subtilisin was engineered to kinetically isolate binding and cleavage reactions. Finally, specific anions were identified to trigger the processing reaction, with fluoride ions being particularly useful. The ability to isolate the binding and processing steps with a triggering mechanism created a protease with a virtual on-off switch. This allowed column-immobilized processing subtilisin to be used as both the affinity ligand and processing protease for one-step purification of proteins. Fusion proteins tagged with the engineered prodomain can be bound to the column and washed free of contaminants. Cleavage can be triggered by the addition of fluoride to release the pure target protein. The column is then regenerated by stripping off the tightly bound prodomain at pH 2.1. Ten proteins have been purified to date by this method.

Combination lithium and divalproex sodium in pediatric bipolarity.

OBJECTIVE: Lithium carbonate (Li) or divalproex sodium (DVPX) may be effective for some juveniles with bipolar disorder. Many youths with bipolar disorder do not respond to DVPX or Li monotherapy. An open-label study was conducted to examine the effectiveness of combination DVPX and Li therapy with youths diagnosed with bipolar disorder. METHOD: Patients meeting DSM-IV criteria for bipolar I or bipolar II disorder, ages 5 to 17 years, were treated prospectively for up to 20 weeks with DVPX + Li. Assessments included the Young Mania Rating Scale (YMRS), Children's Depression Rating Scale-Revised (CDRS-R), and the Children's Global Assessment Scale (CGAS). The a priori definition of clinical remission utilized included four contiguous weekly ratings of YMRS /=51, clinical stability, and no evidence of mood cycling. RESULTS: Ninety patients (66 males, 24 females) were treated. Significant improvement (p <.0001) in all outcome measures was observed by week 8 as well as at the end of study. The mean time in study was 11.3 weeks. Forty-seven percent (n = 42) met a priori criteria for remission. CONCLUSIONS: Symptoms of mania and depression in juvenile bipolar disorder may be safely and effectively treated acutely with DVPX + Li.

Assisting functional assignment for hypothetical Heamophilus influenzae gene products through structural genomics.

The three-dimensional structures of Haemophilus influenzae proteins whose biological functions are unknown are being determined as part of a structural genomics project to ask whether structural information can assist in assigning the functions of proteins. The structures of the hypothetical proteins are being used to guide further studies and narrow the field of such studies for ultimately determining protein function. An outline of the structural genomics methodological approach is provided along with summaries of a number of completed and in progress crystallographic and NMR structure determinations. With more than twenty-five structures determined at this point and with many more in various stages of completion, the results are encouraging in that some level of functional understanding can be deduced from experimentally solved structures. In addition to aiding in functional assignment, this effort is identifying a number of possible new targets for drug development.