W246–W251 Nucleic Acids Research, 2008, Vol. 36, Web Server issue doi:10.1093/nar/gkn259 Published online 14 May 2008 pFlexAna: detecting conformational changes in remotely related proteins Anshul Nigham1,2, Lisa Tucker-Kellogg1,2, Ivana Mihalek3, Chandra Verma3 and David Hsu1,4,* 1Department of Computer Science, National University of Singapore, Singapore 117590, 2Singapore–MIT Alliance, Singapore 117576, 3Bioinformatics Institute (A*STAR), Singapore 138671 and 4Graduate School of Integrative Sciences & Engineering, National University of Singapore, Singapore 117456 Received February 21, 2008; Revised April 11, 2008; Accepted April 20, 2008 ABSTRACT The pFlexAna (protein flexibility analyzer) web server detects and displays conformational changes in remotely related proteins, without relying on sequence homology. To do so, it first applies a reliable statistical test to align core protein frag- ments that are structurally similar and then clusters these aligned fragment pairs into ‘super-alignments’, according to the similarity of geometric transforma- tions that align them. The result is that the dominant conformational changes occur between the clusters, while the smaller conformational changes occur within a cluster. pFlexAna is available at http:// bigbird.comp.nus.edu.sg/pfa2/. INTRODUCTION Conformational change plays a critical role in the func- tioning and regulation of many proteins, and comparing protein structures with diﬀerent backbone conformations is a common task in structural biology (1). This task is particularly challenging when we compare two evolution- arily divergent proteins. The main goal of our work is to provide an automated tool for detecting conformational changes in remotely related proteins. For proteins undergoing conformational change, we can often ﬁnd in them backbone fragments that remain rigid and are well aligned. However, these rigid local fragments reorient themselves with respect to one another during the conformational change, resulting in poor global alignment. A good example is large-scale domain movement. Consider the HECT domain of the human ubiquitin ligase WWP1, which is homologous to the human ubiquitin ligase E6AP. Comparing their crystal structures [PDB codes 1D5F:C (2) and 1ND7 (3)] reveals a dramatic conformational change (Figure 1) crucial for their biological function, which is to move the ubiqui- tin molecule from one substrate protein to another. The crystallographers who determined the WWP1 struc- ture described in detail its comparison with E6AP (3), and they produced several ﬁgures depicting superpositions carried out manually, similar to what pFlexAna produces automatically. To our knowledge, there are currently no other computational tools that aim to automate such analyses. Our pFlexAna (protein ﬂexibility analyzer) web server aims at automatically detecting conformational changes between a pair of protein structures, without relying on sequence homology. Speciﬁcally, it detects regions exhibit- ing structural change, contrasted with regions exhibiting structural similarity. The pFlexAna results can be viewed as indications of the endpoints of hypothesized molecular motions or mutation-induced conformational changes. To detect conformational changes in dissimilar proteins, we must ﬁrst ﬁnd structurally similar fragments (domains, sub-domains, etc.) in the proteins. pFlexAna employs a statistical test to match core protein fragments that are structurally similar and determine the geometric transfor- mations that align them. This statistical test of structural similarity has been shown to be eﬀective in detecting many diﬀerent types of conformational changes (4). pFlexAna displays the protein structures superimposed according to each aligned fragment pair. The superimposed display highlights the discontinuity from the similar to the dis- similar regions of the proteins and aids visualization of the conformational changes that occur relative to the aligned fragment pair used as the basis for superposition. See Figure 3 for a sample result. Furthermore, pFlexAna clusters the aligned fragment pairs into ‘super-alignments’, according to the similarity of geometric transformations that align them. The result is that the dominant conforma- tional changes occur between the clusters, while the smaller conformational changes occur within a cluster. There are many approaches for studying conforma- tional changes. One possibility is to perform molecular dynamics (MD) simulation to generate alternative con- formations from a single known protein structure (5). However, MD simulation is computationally intensive, *To whom correspondence should be addressed. Tel: +65 6516 2978; Fax: +65 6779 4580; Email: dyhsu@comp.nus.edu.sg 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.