Gaohua Liu

A De Novo Protein Binding Pair By Computational Design and Directed Evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.

Preparation of protein samples for NMR structure, function, and small-molecule screening studies.

In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.

High-quality homology models derived from NMR and X-ray structures of E. coli proteins YgdK and Suf E suggest that all members of the YgdK/Suf E protein family are enhancers of cysteine desulfurases

The structural biology of proteins mediating iron‐sulfur (Fe‐S) cluster assembly is central for understanding several important biological processes. Here we present the NMR structure of the 16‐kDa protein YgdK from Escherichia coli, which shares 35% sequence identity with the E. coli protein SufE. The SufE X‐ray crystal structure was solved in parallel with the YdgK NMR structure in the Northeast Structural Genomics (NESG) consortium. Both proteins are (1) key components for Fe‐S metabolism, (2) exhibit the same distinct fold, and (3) belong to a family of at least 70 prokaryotic and eukaryotic sequence homologs. Accurate homology models were calculated for the YgdK/SufE family based on YgdK NMR and SufE crystal structure. Both structural templates contributed equally, exemplifying synergy of NMR and X‐ray crystallography. SufE acts as an enhancer of the cysteine desulfurase activity of SufS by SufE–SufS complex formation. A homology model of CsdA, a desulfurase encoded in the same operon as YgdK, was modeled using the X‐ray structure of SufS as a template. Protein surface and electrostatic complementarities strongly suggest that YgdK and CsdA likewise form a functional two‐component desulfurase complex. Moreover, structural features of YgdK and SufS, which can be linked to their interaction with desulfurases, are conserved in all homology models. It thus appears very likely that all members of the YgdK/SufE family act as enhancers of Suf‐S‐like desulfurases. The present study exemplifies that “refined” selection of two (or more) targets enables high‐quality homology modeling of large protein families.

A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology‐based methods, protein–protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet‐V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution. Proteins 2015; 83:309–317.

NMR and X-RAY structures of human E2-like ubiquitin-fold modifier conjugating enzyme 1 (UFC1) reveal structural and functional conservation in the metazoan UFM1-UBA5-UFC1 ubiquination pathway

For cell regulation, E2-like ubiquitin-fold modifier conjugating enzyme 1 (Ufc1) is involved in the transfer of ubiquitin-fold modifier 1 (Ufm1), a ubiquitin like protein which is activated by E1-like enzyme Uba5, to various target proteins. Thereby, Ufc1 participates in the very recently discovered Ufm1-Uba5-Ufc1 ubiquination pathway which is found in metazoan organisms. The structure of human Ufc1 was solved by using both NMR spectroscopy and X-ray crystallography. The complementary insights obtained with the two techniques provided a unique basis for understanding the function of Ufc1 at atomic resolution. The Ufc1 structure consists of the catalytic core domain conserved in all E2-like enzymes and an additional N-terminal helix. The active site Cys116, which forms a thio-ester bond with Ufm1, is located in a flexible loop that is highly solvent accessible. Based on the Ufc1 and Ufm1 NMR structures, a model could be derived for the Ufc1-Ufm1 complex in which the C-terminal Gly83 of Ufm1 may well form the expected thio-ester with Cys116, suggesting that Ufm1-Ufc1 functions as described for other E1–E2–E3 machineries. α-helix 1 of Ufc1 adopts different conformations in the crystal and in solution, suggesting that this helix plays a key role to mediate specificity.

The NMR solution structure of the 30S ribosomal protein S27e encoded in gene RS27_ARCFU of Archaeoglobus fulgidis reveals a novel protein fold

The Archaeoglobus fulgidis gene RS27_ARCFU encodes the 30S ribosomal protein S27e. Here, we present the high‐quality NMR solution structure of this archaeal protein, which comprises a C4 zinc finger motif of the CX2CX14‐16CX2C class. S27e was selected as a target of the Northeast Structural Genomics Consortium (target ID: GR2), and its three‐dimensional structure is the first representative of a family of more than 116 homologous proteins occurring in eukaryotic and archaeal cells. As a salient feature of its molecular architecture, S27e exhibits a β‐sandwich consisting of two three‐stranded sheets with topology B(↓), A(↑), F(↓), and C(↑), D(↓), E(↑). Due to the uniqueness of the arrangement of the strands, the resulting fold was found to be novel. Residues that are highly conserved among the S27 proteins allowed identification of a structural motif of putative functional importance; a conserved hydrophobic patch may well play a pivotal role for functioning of S27 proteins, be it in archaeal or eukaryotic cells. The structure of human S27, which possesses a 26‐residue amino‐terminal extension when compared with the archaeal S27e, was modeled on the basis of two structural templates, S27e for the carboxy‐terminal core and the amino‐terminal segment of the archaeal ribosomal protein L37Ae for the extension. Remarkably, the electrostatic surface properties of archaeal and human proteins are predicted to be entirely different, pointing at either functional variations among archaeal and eukaryotic S27 proteins, or, assuming that the function remained invariant, to a concerted evolutionary change of the surface potential of proteins interacting with S27.

High-Quality NMR Structure of Human Anti-Apoptotic Protein Domain Mcl-1(171-327) for Cancer Drug Design.

A high-quality NMR solution structure is presented for protein hMcl-1(171–327) which comprises residues 171–327 of the human anti-apoptotic protein Mcl-1 (hMcl-1). Since this construct contains the three Bcl-2 homology (BH) sequence motifs which participate in forming a binding site for inhibitors of hMcl-1, it is deemed to be crucial for structure-based design of novel anti-cancer drugs blocking the Mcl1 related anti-apoptotic pathway. While the coordinates of an NMR solution structure for a corresponding construct of the mouse homologue (mMcl-1) are publicly available, our structure is the first atomic resolution structure reported for the ‘apo form’ of the human protein. Comparison of the two structures reveals that hMcl-1(171–327) exhibits a somewhat wider ligand/inhibitor binding groove as well as a different charge distribution within the BH3 binding groove. These findings strongly suggest that the availability of the human structure is of critical importance to support future design of cancer drugs.

NMR structure of protein yjbR from Escherichia coli reveals ‘double-wing’ DNA binding motif

Kiran Kumar Singarapu, Gaohua Liu, Rong Xiao, Claudia Bertonati, Barry Honig, Gaetano T. Montelione, and Thomas Szyperski* Department of Chemistry, State University of New York at Buffalo, Buffalo, New York Northeast Structural Genomics Consortium Center of Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York

NMR structure of the hypothetical protein AQ-1857 encoded by the Y157 gene from Aquifex aeolicus reveals a novel protein fold.

Duanxiang Xu, Gaohua Liu, Rong Xiao, Tom Acton, Sharon Goldsmith-Fischman, Barry Honig, Gaetano T. Montelione, and Thomas Szyperski* Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York Center of Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York Northeast Structural Genomics Consortium

NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis reveals a distinct 5-helix bundle.

Gaohua Liu, Dinesh K. Sukumaran, Duanxiang Xu, Yiwen Chiang, Thomas Acton, Sharon Goldsmith-Fischman, Barry Honig, Gaetano T. Montelione, and Thomas Szyperski* Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, New York Center of Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York Northeast Structural Genomics Consortium